U.S. patent number 9,572,863 [Application Number 14/364,449] was granted by the patent office on 2017-02-21 for methods for preventing or treating certain disorders by inhibiting binding of il-4 and/or il-13 to their respective receptors.
This patent grant is currently assigned to Pieris Pharmaceuticals GmbH. The grantee listed for this patent is PIERIS PHARMACEUTICALS GMBH. Invention is credited to Laurent Audoly, Andreas Hohlbaum, Beverly Koller.
United States Patent |
9,572,863 |
Hohlbaum , et al. |
February 21, 2017 |
Methods for preventing or treating certain disorders by inhibiting
binding of IL-4 and/or IL-13 to their respective receptors
Abstract
The present disclosure relates to methods of treating,
ameliorating or preventing a disorder comprising administering a
therapeutically effective amount of a composition comprising a
protein which inhibits IL-4 and/or IL-13 from binding to their
respective receptors to a subject in need thereof. In some
embodiments, the disorder is preferably associated with an increase
of the Th2 immune response. In some embodiments, administration is
preferably locally to the lung in order to treat, ameliorate or
prevent allergic asthma, rhinitis, conjunctivitis, lung fibrosis,
cystic fibrosis, chronic obstructive pulmonary disease, pulmonary
alveolar proteinosis or adult respiratory distress syndrome.
Inventors: |
Hohlbaum; Andreas (Paunzhausen,
DE), Audoly; Laurent (Mahwah, NJ), Koller;
Beverly (Chapel Hill, NC) |
Applicant: |
Name |
City |
State |
Country |
Type |
PIERIS PHARMACEUTICALS GMBH |
Freising-Weihenstephan |
N/A |
DE |
|
|
Assignee: |
Pieris Pharmaceuticals GmbH
(Freising-Weihenstephan, DE)
|
Family
ID: |
47552959 |
Appl.
No.: |
14/364,449 |
Filed: |
December 12, 2012 |
PCT
Filed: |
December 12, 2012 |
PCT No.: |
PCT/EP2012/075146 |
371(c)(1),(2),(4) Date: |
June 11, 2014 |
PCT
Pub. No.: |
WO2013/087660 |
PCT
Pub. Date: |
June 20, 2013 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20140357548 A1 |
Dec 4, 2014 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
61570018 |
Dec 13, 2011 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
9/0078 (20130101); A61K 9/08 (20130101); A61P
11/00 (20180101); A61P 11/02 (20180101); A61P
35/00 (20180101); A61K 38/1709 (20130101); A61P
11/16 (20180101); A61P 11/06 (20180101); A61P
29/00 (20180101); A61P 27/02 (20180101); A61P
43/00 (20180101); A61P 37/08 (20180101) |
Current International
Class: |
A61K
38/00 (20060101); A61K 38/17 (20060101); A61K
9/00 (20060101); A61K 9/08 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
2009-545301 |
|
Dec 2009 |
|
JP |
|
WO 2008/015239 |
|
Feb 2008 |
|
WO |
|
WO 2008015239 |
|
Feb 2008 |
|
WO |
|
WO 2011/154420 |
|
Dec 2011 |
|
WO |
|
Other References
Davidson et al., Genetics and pulmonary medicine. THORAX. 1998;
53:389-397. cited by examiner .
Holloway et al. Genetics of allergic disease. J Allergy Olin
Immunol. vol. 125, No. 2, Feb. 2010, pp. S81-S94. cited by examiner
.
Cowden et al., "Histamine H4 receptor antagonism diminishes
existing airway inflammation and dysfunction via modulation of Th2
cytokines," Respiratory Research, 11:86, Jun. 2010 (12 pages).
cited by applicant .
European Office Action dated Jul. 10, 2015 issued in Application
No. 12813295.8. cited by applicant .
Fiset et al., "Modulation of allergic response in nasal mucosa by
antisense oligodeoxynucleotides for IL-4," Journal of Allergy and
Clinical Immunology, 111:580-586, Jan. 2003. cited by applicant
.
Moore et al, "IL-13 and IL-4 cause eotaxin release in human airway
smooth muscle cells: a role for ERK," American Journal of
Physiology. Lung Cellular and Molecular Physiology, 282:L847-L853,
Nov. 2001. cited by applicant .
Terada et al., "Contribution of IL-18 to atopic-dermatitis like
skin inflammation induced by Staphylococcus aureus product in
mice," PNAS, 103:8616-8821, Jun. 2006. cited by applicant .
Walker et al., "RNA interference of State rapidly attenuates
ongoing inter-leukin-13-mediated events in lung epithelial cells,"
Immunology, 127:256-266, Jun. 2009. cited by applicant .
Loefblom et al., "Non-immunoglobulin based protein scaffolds,"
Current Opinion in Biotechnology, 2011, 22:843-848. cited by
applicant .
Hohibaurn et al., "Next generation therapeutics for the treatment
of respiratory diseases--discovery and characterization of an
inhalable highly potent and specific anti-IL-4RA small protein
antagonist," Inflamm. Res., Jun. 2011, 60(Supp1):82-83, 10.sup.th
World Congress on Inflammation, Paris, France, Jun. 25-29, 2011.
cited by applicant .
Hirst, Stuart J., et al.; "Selective Induction of Eotaxin Release
by Interleukin-3 or Interleukin-4 in Human Airway Smooth Muscle
Cells is Synergistic with Interleukin-1.beta. and is Mediated by
the Interleukin-4 Receptor .alpha.--Chain"; American Journal of
Respiratory and Critical Care Medicine, vol. 165, No. 8, pp.
1161-1171, 2002. cited by applicant .
Japan Patent Office Notice of Reasons for Rejection issued in
application 2014-546471 dated May 23, 2016; pp. 1-5 (with English
translation). cited by applicant.
|
Primary Examiner: Lieb; Jeanette
Attorney, Agent or Firm: Foley & Lardner LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a National Stage application of
PCT/EP2012/075146, filed Dec. 12, 2012, which claims priority from
U.S. Provisional Application 61/570,018, filed Dec. 13, 2011.
Claims
The invention claimed is:
1. A method of treating or ameliorating a lung disorder that is
related to the immune system, comprising administering to a subject
in need thereof a human tear lipocalin mutein or a fusion protein,
conjugate or composition thereof that is capable of inhibiting IL-4
and/or IL-13 from binding to their respective receptors, wherein
said lipocalin mutein is capable of exhibiting in vivo therapeutic
activity in the subject, wherein said lipocalin mutein has at least
75% sequence identity to the amino acid sequence of mature human
tear lipocalin (SEQ ID NO: 1), wherein said lipocalin mutein has
the following amino acid residues at amino acid positions
corresponding to numbering of amino acids positions of mature human
tear lipocalin (SEQ ID NO: 1): Ser at amino acid position 26, Arg
at amino acid position 27, Cys at amino acid position 28, Arg at
amino acid position 30, Ala at amino acid position 31, Val at amino
acid position 32, Tyr at amino acid position 33, Asn at amino acid
position 34, Phe at amino acid position 53, Ala at amino acid
position 55, Gln at amino acid position 56, Arg at amino acid
position 57, Lys at amino acid position 58, Trp at amino acid
position 61, Lys at amino acid position 63, Tyr at amino acid
position 64, Leu at amino acid position 66, Ser at amino acid
position 80, Arg at amino acid position 83, Leu at amino acid
position 104, Cys at amino acid position 105, Pro at amino acid
position 106, and Gln at amino acid position 108.
2. The method of claim 1, wherein said lipocalin mutein is capable
of disrupting downstream signaling and/or cellular responses
induced by IL-4 and/or IL-13.
3. The method of claim 1, wherein said lipocalin mutein has the
amino acid sequence as set forth in SEQ ID NO: 6.
4. The method of claim 1, wherein said lipocalin mutein is capable
of inhibiting in vivo human IL-13 induced transcript Ccl11
(eotaxin).
5. The method of claim 1, wherein said lipocalin mutein is more
potent than an IL-4 mutant (R121D, Y124D) when inhibiting human
IL-13 induced transcript Ccl11 (eotaxin) in vivo.
6. The method of claim 1, wherein said lipocalin mutein is capable
of exhibiting a functional activity as good as an anti-IL-4RA
monoclonal antibody AMG 317 having the variable light and heavy
chain sequence as shown in SEQ ID NO: 14 and 15, respectively,
wherein said lipocalin mutein is capable of inhibiting
IL-13-induced goblet cell metaplasia as good as the anti-IL-4RA
monoclonal antibody.
7. The method of claim 1, wherein said lipocalin mutein, fusion
protein, conjugate or composition is locally administered to the
lung.
8. The method of claim 7, wherein said lipocalin mutein, fusion
protein, conjugate or composition is administered via aerosol
inhalation.
9. The method of claim 1, wherein said lipocalin mutein, fusion
protein, conjugate or composition is administered to a subject in
need thereof at a frequency selected from the group consisting of:
up to four times daily, up to three times daily, up to twice daily,
up to once daily, up to once every other day, up to once every
third day, up to once every week, and up to once every other
week.
10. The method of claim 1, wherein said lipocalin mutein, fusion
protein, conjugate or composition is administered to a subject in
need thereof every time at a dosage level selected from the group
consisting of: 0.06-600 mg, 0.06-100 mg, 0.3-10 mg, and 1-3 mg.
11. The method of claim 1, wherein said disorder is associated with
an increase of the Th2 immune response, an allergic reaction, an
allergic inflammation, a mucus production, or a mucus
secretion.
12. The method of claim 1, wherein the composition further
comprises a pharmaceutically acceptable formulation.
13. The method of claim 1, wherein said composition further
comprises an anti-allergic medicament, an anti-allergic
inflammation medicament and/or an anti-mucus medicament.
14. The method of claim 1, wherein said lipocalin mutein is
cross-reactive with marmoset IL-4RA and is not cross-reactive with
mouse IL-4RA and/or cynomolgus IL-4RA.
15. The method of claim 1, wherein said lipocalin mutein is
essentially not cross-reactive with IL-23 receptor alpha chain,
type I cytokine receptor with fibronectin type III domain, IL-6
receptor or IL-18 receptor alpha chain.
16. The method of claim 1, wherein the disorder is selected from
the group consisting of asthma, allergic asthma, rhinitis, lung
fibrosis, and adult respiratory distress syndrome.
17. The method of claim 1, wherein said lipocalin mutein has at
least 90% sequence identity to the amino acid sequence set forth in
SEQ ID NO:6.
18. The method of claim 1, wherein said lipocalin mutein has at
least 95% sequence identity to the amino acid sequence set forth in
SEQ ID NO:6.
19. The method of claim 3, wherein the disorder is selected from
the group consisting of asthma, allergic asthma, rhinitis, lung
fibrosis, and adult respiratory distress syndrome.
20. The method of claim 19, wherein the disorder is asthma.
Description
The instant application contains a Sequence Listing which has been
submitted in ASCII format via EFS-WEB and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Aug. 12,
2014, is named sequence.txt and is 30 KB.
FIELD OF THE INVENTION
The present disclosure relates to methods of treating, ameliorating
or preventing a disorder comprising administering a therapeutically
effective amount of a composition comprising a protein which
inhibits the ligand of Uniprot #P05112 and/or the ligand of Uniprot
#P35225 from binding to their respective receptors to a subject in
need thereof. In some embodiments, the disorder is preferably
associated with an increase of the Th2 immune response. In some
embodiments, administration is preferably locally to the lung in
order to treat, ameliorate or prevent allergic asthma, rhinitis,
conjunctivitis, lung fibrosis, cystic fibrosis, chronic obstructive
pulmonary disease, pulmonary alveolar proteinosis or adult
respiratory distress syndrome.
BACKGROUND
Proteins that selectively bind to selected targets by way of
non-covalent interaction play a crucial role as reagents in
biotechnology, medicine, bioanalytics as well as in the biological
and life sciences in general. Antibodies, i.e. immunoglobulins, are
a prominent example of this class of proteins. Despite the manifold
needs for such proteins in conjunction with recognition, binding
and/or separation of ligands/targets, almost exclusively
immunoglobulins are currently used.
Additional proteinaceous binding molecules that have antibody-like
functions are the members of the lipocalin family, which have
naturally evolved to bind ligands. Lipocalins occur in many
organisms, including vertebrates, insects, plants and bacteria. The
members of the lipocalin protein family (Pervaiz, S., & Brew,
K. (1987) FASEB J. 1, 209-214) are typically small, secreted
proteins and have a single polypeptide chain. They are
characterized by a range of different molecular-recognition
properties: their ability to bind various, principally hydrophobic
molecules (such as retinoids, fatty acids, cholesterols,
prostaglandins, biliverdins, pheromones, tastants, and odorants),
their binding to specific cell-surface receptors and their
formation of macromolecular complexes. Although they have, in the
past, been classified primarily as transport proteins, it is now
clear that the lipocalins fulfill a variety of physiological
functions. These include roles in retinol transport, olfaction,
pheromone signaling, and the synthesis of prostaglandins. The
lipocalins have also been implicated in the regulation of the
immune response and the mediation of cell homoeostasis (reviewed,
for example, in Flower, D. R. (1996) Biochem. J. 318, 1-14 and
Flower, D. R. et al. (2000) Biochim. Biophys. Acta 1482, 9-24).
The lipocalins share unusually low levels of overall sequence
conservation, often with sequence identities of less than 20%. In
strong contrast, their overall folding pattern is highly conserved.
The central part of the lipocalin structure consists of a single
eight-stranded anti-parallel .beta.-sheet closed back on itself to
form a continuously hydrogen-bonded .beta.-barrel. This
.beta.-barrel forms a central cavity. One end of the barrel is
sterically blocked by the N-terminal peptide segment that runs
across its bottom as well as three peptide loops connecting the
.beta.-strands. The other end of the .beta.-barrel is open to the
solvent and encompasses a target-binding site, which is formed by
four flexible peptide loops. It is this diversity of the loops in
the otherwise rigid lipocalin scaffold that gives rise to a variety
of different binding modes each capable of accommodating targets of
different size, shape, and chemical character (reviewed, e.g., in
Flower, D. R. (1996), supra; Flower, D. R. et al. (2000), supra, or
Skerra, A. (2000) Biochim. Biophys. Acta 1482, 337-350).
Various PCT publications (e.g., WO 99/16873, WO 00/75308, WO
03/029463, WO 03/029471 and WO 2005/19256) disclose how muteins of
various lipocalins (e.g. tear lipocalin and hNGAL lipocalin) can be
constructed to exhibit a high affinity and specificity against a
target that is different than a natural ligand of a wild type
lipocalin. This can be done, for example, by mutating one or more
amino acid positions of at least one of the four peptide loops. In
addition, PCT publication WO 2011/154420 teaches one or more
methods for producing lipocalin muteins, which bind to IL-4
receptor subunit alpha.
Th2 cytokines IL-4 (officially known as Interleukin-4, Uniprot
#P05112) and IL-13 (officially known as Interleukin-13, Uniprot
#P35225) have largely overlapping functions and directly promote
several key features of asthma including eosinophilia, goblet cell
metaplasia, airway hyperresponisveness, IgE immunoglobin switch,
alternative macrophage activation, smooth muscle cell remodeling
and subepithelial fibrosis. Furthermore, genetic polymorphism in
the genes of IL-4, IL-13, IL-4RA (officially known as Interleukin-4
receptor subunit alpha, Uniprot #P24394, SEQ ID NO: 12) and Stat6
are linked with Asthma. This is particular relevant as the
combination of allelic variant of IL-4, IL-13, IL-4RA and Stat6
appear synergistic and described polymorphism in IL-4, IL-13 and
IL-4RA enhance the production, function or signaling activity of
the Th2 cytokines or common subunit of the IL-4/IL-13 receptor
(Finkelman et al. JI, 2010, 184:1663-74). Recently, asthma
endotypes or subphenotypes have been defined by molecular mechanism
or treatment response. Woodruff et al for example defined asthma
phenotypes into Th2-high and Th2-low based on the lung epithelial
expression of IL-13-inducible genes POSTN (periostin), CLCA1 (gobs)
and SERPINB2 (Wooddruff P G et al, Genome-wide profiling identifies
epithelial cell genes associated with asthma and with treatment
response to corticosteroids PNAS, 2007, 104: 15858-63; Prescott G.
Woodruff) (T-helper Type 2-driven inflammation defines major
subphenotypes of Asthma, Am. J. Respir. Crti. Care Med., 2009,
180:388-395).
Therefore, it would be desirable to have improved therapeutic
methods involving therapeutically effective amount of a composition
comprising muteins of human tear lipocalin, binding to IL-4RA with
high binding affinity and therefore inhibiting IL-4 and/or IL-13
from binding to their respective receptors, that exhibit in vivo
therapeutic activities in a subject in need thereof. When used in
the present application, a subject in need thereof may be a mammal,
such as a human, a dog, a mouse, a rat, a pig, an ape such as
cynomolgus to name only a few illustrative examples, which is in
need of a treatment or prevention of a disorder.
SUMMARY OF THE DISCLOSURE
The present disclosure relates to a method of treating,
ameliorating or preventing a disorder comprising administering a
therapeutically effective amount of a composition to a subject in
need of such composition, wherein said composition comprises a
lipocalin mutein of the disclosure that is capable of inhibiting
IL-4 and/or IL-13 from binding to their respective receptors. In
some further embodiment, the lipocalin mutein is capable of
disrupting downstream signaling and/or cellular responses induced
by IL-4 and/or IL-13. In various embodiments, the disorder is a
disorder in which the IL4/IL13 pathway contributes to disease
pathogenesis. In various further embodiments, the composition may
be locally administered to the lung. In various preferred
embodiments, the composition may be administered via aerosol
inhalation. In some still preferred embodiments, the composition is
administered to a subject in need thereof at a frequency selected
from the group consisting of: up to four times daily, up to three
times daily, up to twice daily, up to once daily, up to once every
other day, up to once every third day, up to once every week and up
to once every other week.
In some embodiments, when used in the present application, a
subject in need of such composition is suffering from a disorder in
which the IL-4 expression and/or IL-13 expression contributes or is
related to disease pathogenesis or aggravation. In some
embodiments, the subject, in need of such composition, is suffering
from a disorder that can be improved, ameliorated or inhibited by
removal, inhibition or reduction of the IL-4 activity and/or IL-13
activity. In some further embodiments, a subject in need of such
composition may be suffering from one or more disorders including,
for example, allergic inflammation, allergic asthma, rhinitis,
conjunctivitis, lung fibrosis, cystic fibrosis, chronic obstructive
pulmonary disease, pulmonary alveolar proteinosis or adult
respiratory distress syndrome. It is also envisaged that the
lipocalin mutein of the disclosure, in some embodiments, is applied
for the treatment, amelioration or prevention of tissue fibrosis
(see Chiaramonte et al. (1999), J. Clin. Invest. 104(6), 777-785.
The fibrosis may preferably result from healing of a wound, for
example, a wound from a surgical incision. The tissue fibrosis
affects, for example, a tissue selected from the group consisting
of liver, skin epidermis, skin endodermis, muscle, tendon,
cartilage, cardiac tissue, pancreatic tissue, lung tissue, uterine
tissue, neural tissue, testis, ovary, adrenal gland, artery, vein,
colon, small intestine, biliary tract and gut, in particular, the
tissue being selected from lung and liver.
For example, one disorder that can be preferably treated,
ameliorated or prevented by a method of the disclosure is
associated with an increase of the Th2 immune response.
In some embodiments, the disorder can be associated with an
allergic reaction or an allergic inflammation, preferably, the
allergic reaction is a food allergy, and preferably, the allergic
inflammation is associated with allergic asthma, rhinitis,
conjunctivitis or dermatitis. In various preferred embodiments, the
allergic asthma can be an airway inflammation in which the IL4/IL13
pathway contributes to disease pathogenesis.
In other preferred embodiments, the composition of the disclosure
further comprises an anti-allergic medicament and/or an
anti-allergic inflammation medicament.
Another exemplary disorder that can be preferably treated,
ameliorated or prevented by a method of the disclosure is
associated with a mucus production or a mucus secretion.
In various preferred embodiments, the disorder can be a lung
disorder, and preferably a chronic obstructive pulmonary disease
(COPD) or cystic fibrosis (CF).
In a still further embodiment, the said composition can be used as
a gene therapy agent for various disclosed disorders.
A lipocalin mutein of the disclosure can be a human tear lipocalin
mutein which has at any one or more amino acids at a position
corresponding to position 27, 28, 30, 31, 33, 53, 57, 61, 64, 66,
80, 83, 104-106 and 108 of the linear polypeptide sequence of the
mature human tear lipocalin a mutated amino acid. However, the
lipocalin mutein of the disclosure can also be a mutein of a
lipocalin other than human tear lipocalin, such as human NGAL
lipocalin or other lipocalins described herein.
In various preferred embodiments, the lipocalin mutein has at any
two or more amino acids at a position corresponding to position 26,
32, 34, 55, 56, 58 and 63 of the linear polypeptide sequence of the
mature human tear lipocalin a mutated amino acid.
The lipocalin mutein described herein has in a particularly
preferred embodiment at least 75% identity to the sequence of
mature human tear lipocalin (SEQ ID NO: 1).
A lipocalin mutein applied in the methods of the present disclosure
is preferably cross-reactive with another species, e.g. marmoset
IL-4RA, which allows testing of the then-candidate lipocalin mutein
in marmoset apes resembling the human organism. The lipocalin
mutein may or may not be cross-reactive with IL-4RA from other
non-human species such as mouse IL-4RA and/or cynomolgus
IL-4RA.
It is generally preferred that the lipocalin mutein is essentially
not cross-reactive with a related protein or non-related protein.
Said related protein is, for example, an IL-23 receptor alpha chain
and/or type I cytokine receptor with fibronectin type III domain,
while said non-related protein is, for example, an IL-6 receptor
and/or IL-18 receptor alpha chain.
DESCRIPTION OF THE FIGURES
FIG. 1 depicts inhibition of human IL-4 and IL-13 induced STAT 6
phosphorylation (FACS-based assay) in TF-1 cells by a lipocalin
mutein of the disclosure.
FIG. 2 depicts inhibition of human IL-4 induced STAT 6
phosphorylation (FACS-based assay) in human IL-4RA/IL-13RA1
(officially known as Interleukin-13 receptor subunit alpha-1,
Uniprot #P78552, SEQ ID NO: 13) chain double knock-in mice by a
lipocalin mutein of the disclosure.
FIG. 3 depicts inhibition of human IL-13 induced eotaxin in lung
tissue of human IL-4 receptor alpha/IL-13 receptor alpha 1 chain
double knock-in mice by a lipocalin mutein of the disclosure.
FIG. 4 depicts inhibition of human IL-13 induced eotaxin transcript
in human IL-4 receptor alpha/IL-13 receptor alpha 1 chain double
knock-in mice by a lipocalin mutein of the disclosure when
administered at different time intervals (FIG. 4a) or different
doses (FIG. 4b), prior to administration of human IL-13, as well as
in comparison with an IL-4 mutant at different dose levels (FIG.
4c).
FIG. 5 depicts the analysis of a nebulized lipocalin mutein by size
exclusion chromatography and laser diffraction.
FIG. 6 depicts the pharmacokinetic properties and biodistribution
of a lipocalin mutein of the disclosure in human IL-4RA/human IL-13
double knock-in mice after intratracheal instillation.
FIG. 7 depicts the inhibition of IL-13 induced goblet cell
metaplasia in human airway epithelium air-liquid-interface culture
system by a lipocalin mutein of the disclosure.
DETAILED DESCRIPTION
The present disclosure provides a method of treating, ameliorating
or preventing a disclosed disorder, comprising administering a
therapeutically effective amount of a composition comprising a
lipocalin mutein, which inhibits IL-4 and/or IL-13 from binding to
their respective receptors by binding to IL-4 receptor alpha chain,
to a subject in need thereof. A lipocalin mutein for use in
accordance with the present disclosure can specifically bind to
IL-4 receptor alpha chain. Advantageously, these lipocalin muteins
have a high binding affinity for the IL-4 receptor alpha chain.
These lipocalin muteins have even improved binding properties for
IL-4 receptor alpha in relation to the lipocalin muteins provided
in WO 2008/015239; in particular, they have a higher binding
affinity.
The present inventors have demonstrated that lipocalin muteins of
the present disclosure, due to their binding of the IL-4 receptor
alpha chain, interfere with the interaction of the receptors'
cognate ligands, i.e., the cytokine(s) IL-4 and/or IL-13, in a
manner to bring about a therapeutic response in vivo. For example,
data from a transgenic mouse model, which was used to investigate
effects that lipocalin muteins of the disclosure may have on IL-4
and/or IL-13 mediated signaling, demonstrate a disruption of
downstream signaling mediated by IL-4 and IL-13. The transgenic
mouse, which resembles a human in that the mice carry genes
encoding the human IL-4 receptor alpha chain and the IL-13 receptor
alpha 1 chain, are representative of the therapeutic potential in
humans. To this end, the human genes are located in their
corresponding locus of the respective mouse chromosome, thereby
rendering the mouse to be a double knock-in mouse expressing both
the type I and type II receptor. Therefore, when used herein a
"therapeutically effective amount" is preferably an amount of a
lipocalin mutein of the present disclosure that is therapeutically
effective in a human.
In contrast with Examples of the disclosure, the prior art does not
teach the skilled person whether a lipocalin mutein of the
disclosure is capable of is capable of disrupting downstream
signaling or cellular responses induced by IL-4 and/or IL-13 in
subjects, and/or capable of exhibiting an in vivo therapeutic
activity in subjects suffering from one or more disorders, such as,
allergic inflammation, allergic asthma, rhinitis, conjunctivitis,
lung fibrosis, cystic fibrosis, chronic obstructive pulmonary
disease, pulmonary alveolar proteinosis or adult respiratory
distress syndrome. Nor can any information about the effectiveness
of lipocalin muteins against these disorders be derived from the
prior art. In the absence of any data demonstrating a therapeutic
effect of a lipocalin mutein against disorders, for instance,
associated with an increase of the Th2 immune response and/or with
an allergic reaction or an allergic inflammation, such therapeutic
effect and corresponding methods of treatment, prevention or
amelioration could not reasonably have been expected.
Indeed, the present disclosure provides in vivo data showing a
therapeutic effect of the lipocalin muteins described herein which
illustrate, for example, the anti-inflammatory effect of the
lipocalin muteins of the present disclosure. In this regard, the
present disclosure shows, for the first time, that a lipocalin
mutein of the disclosure (SEQ ID NO: 6) is capable of inhibiting
human IL-13 induced transcript Ccl11 (eotaxin) effectively in vivo
and is more potent than an IL-4 mutant (the IL-4 (R121D, Y124D) in
FIG. 4c) as measured by a test essentially described in Example 5:
prepare several human IL-4 receptor alpha/human IL-13 receptor
alpha chain 1 double knock-in mice as described in Example 4 with
the exception that 30 .mu.l of IL-13 (Peprotech, 1 .mu.g) was
administered by intratracheal instillation only once, optionally,
set the test parameters as described in Example 5 and group the
mice accordingly, apply the lipocalin mutein (SEQ ID NO: 6) by
intratracheal instillation with a volume of 30 .mu.l either at a
constant dose of 98 .mu.g at different times prior to the IL-13
dose or at different amounts 30 minutes prior to the IL-13 dose,
apply the IL-4 mutant (R121D, Y124D) also by intratracheal
instillation with a volume of 30 .mu.l at different amounts 30
minutes prior to the IL-13 dose, use the abbreviated IL-13 induced
airway inflammation model (single IL-13 intratracheal
administration) to assess the duration of the pharmacological
response, dose dependency and comparable potency. The therapeutic
effect observed on the murine animal model applied in the present
disclosure thus provides sufficient evidence of a therapeutic
application, given the presence of human orthologues and absence of
murine orthologues. Based upon the this principle, it is more than
reasonable that, in the absence of any data on human patients, the
in vivo experiment are sufficiently predictive of the in vivo
activity, for example the in vivo anti-inflammatory activity, of
the lipocalin muteins described herein in humans.
In addition, the present disclosure demonstrates that a lipocalin
mutein of the disclosure (SEQ ID NO: 6) is capable of exhibiting a
functional activity as good as an anti-IL-4RA monoclonal antibody
(the anti-IL4R mAb in FIG. 7; the light and heavy chain variable
region of which are shown in SEQ ID NOs: 14 and 15, respectively)
disclosed in Example 8, as measured by a test essentially described
in Example 8: treat MucilAir.TM. (an air-liquid interface culture
system with human airway epithelium reconstituted in vitro using
primary human cells) every two days with human IL-13 at about 0.3
to 30 ng/ml, optionally, perform an in-situ Alcain blue stain
(stains acid mucopolysacharides and glycosaminoglycans in blue to
bluish-green), and/or perform histological analysis, in order to
demonstrate that MucilAir.TM. shows an increased goblet cell
density after about 14 days of treatment, test the inhibitory
effect of a lipocalin mutein on goblet cell metaplasia by comparing
continuous exposure of MucilAir.TM. to about 10 ng/ml human IL-13
for 14 days as positive control, to IL-13 plus different
concentrations of a lipocalin mutein, to IL-13 plus different
concentrations of said anti-IL-4RA monoclonal antibody (the light
and heavy chain variable region are shown in SEQ D NOs: 14 and 15),
and to MucilAir.TM. cultured for 14 days without IL-13 as negative
control, perform an Alcain blue stain, e.g., dd Alcain blue stain
to the apical surface for an in-situ stain and take pictures from
stained cells under a phase contrast microscope for image analysis,
quantify the percentage of the Alcain blue positive cells by the
public domain Java image processing program ImageJ and express
their number as area ratio of Alcain blue area/total image area,
optionally, measure eotaxin-3 (an IL-13 induced chemokine) in the
basal medium on day 14 using a commercially available
ultrasensitive eotaxin-3 Kit from, e.g. Meso Scale Discovery. Said
anti-IL-4RA antibody has the variable light and heavy chain regions
of SEQ ID NOs: 14 and 15, respectively, and was developed by Amgen
(former Immunex) and is called AMG 317. It is a fully human
monoclonal antibody that was under investigation for its ability to
block the actions of interleukin-4 and interleukin-13 that play a
role in asthma (in 2008, a phase 2 dose ranging study in moderate
to severe asthma was completed). An interim analysis showed
evidence of biological activity; however, the overall clinical
efficacy did not meet the expectations. Thus, the lipocalin mutein
of SEQ ID NO: 6 is capable of inhibiting IL-13 induced goblet cell
metaplasia as good as the anti-IL-4RA monoclonal antibody described
herein, when the comparison of the lipocalin mutein with said
anti-IL-4RA antibody was done as described above. It is preferred
in some further embodiments, therefore, that a lipocalin mutein of
the disclosure has in the above-described test the same properties
as the lipocalin mutein of SEQ ID NO: 6 with respect to the
inhibition of goblet cell metaplasia.
The IL-4/IL-4R.alpha. complex can dimerize with either the common
gamma chain (.gamma.c, CD132) or the IL-13Ralpha1 (IL-13R.alpha.1)
subunit, via domains on IL-4, to create two different signaling
complexes, commonly referred to as Type I and Type II receptors,
respectively. Alternatively, IL-13 can bind IL-13R.alpha.1 to form
an IL-13/IL-13R.alpha.1 complex that recruits the IL-4R.alpha.
subunit to form a Type II receptor complex. Thus, IL-4R.alpha.
mediates the biological activities of both IL-4 and IL-13 (reviewed
by Gessner et al, Immunobiology, 201:285, 2000). The lipocalin
muteins of the present disclosure advantageously interfere with
and/or block the signaling via the Type I and/or Type II receptors,
since these lipocalin muteins are capable of binding to the IL-4
receptor alpha chain.
In vitro studies have shown that IL-4 and IL-13 activate effector
functions in a number of cell types, for example in T cells, B
cells, eosinophils, mast cells, basophils, airway smooth muscle
cells, respiratory epithelial cells, lung fibroblasts, and
endothelial cells (reviewed by Steinke et al, Resp Res, 2:66, 2001,
and by Willis-Karp, Immunol Rev, 202:175, 2004). IL-4R.alpha. is
expressed in low numbers (100-5000 molecules/cell) on a variety of
cell types (Lowenthal et al, J Immunol, 140:456, 1988), e.g.
peripheral blood T cells, monocytes, airway epithelial cells, B
cells and lung fibroblasts. The type I receptor predominates in
hematopoietic cells, whereas the type II receptor is expressed on
both hematopoietic cells and non-hematopoietic cells.
The cell surface receptors and receptor complexes bind IL-4 and/or
IL-13 with different affinities. The principal components of
receptors and receptor complexes that bind IL-4 and/or IL-13 are
IL-4R.alpha., IL-13R.alpha.1 and IL-13R.alpha.2. These chains are
expressed on the surface of cells as monomers or heterodimers of
IL-4R.alpha./IL-13R.alpha.1 or IL-4R.alpha./IL-13R.alpha.2.
IL-4-r.alpha. monomer binds IL-4, but not IL-13. IL-13R.alpha.1 and
IL-13R.alpha.2 monomers bind IL-13, but do not bind IL-4.
IL-4R.alpha./IL-13R.alpha.1 and IL-4R.alpha./IL-13R.alpha.2
heterodimers bind both IL-4 and IL-13. Given the fact that the
IL-13 receptor alpha 2 (IL-13Ralpha2) that binds IL-13 with high
affinity, but is a non-signaling receptor which is believed to be a
decoy receptor for IL-13, it is envisaged that the lipocalin
muteins of the present disclosure do preferably not bind to the
IL-13 receptor alpha 2.
As explained, both IL-4 and IL-13 signal via the IL-4R.alpha., a
component of the type I (IL-4R.alpha. and .gamma.c) and type II
receptors (IL-4R.alpha. and IL-13R.alpha.1). IL-4 signals via both
type I and II receptor pathways, whereas IL-13 signals only via the
type II IL-4R. IL-13 also binds to the IL-13R.alpha.2 chain, which
does not contain a trans-membrane-signaling domain and is thought
to act as a decoy receptor. .gamma.c activates Janus kinase (JAK)3,
whereas IL-13R.alpha.1 activates tyrosine kinase 2 (TYK2) and JAK2.
Activated JAKs then phosphorylate STAT-6. Phosphorylated STAT-6
dimerizes, migrates to the nucleus, and binds to the promoters of
the IL-4 and IL-13 responsive genes, such as those associated with
T-helper type 2 (Th2) cell differentiation, airway inflammation,
airway hyper-responsiveness (AHR) and mucus production.
Th2-type immune responses promote antibody production and humoral
immunity, and are elaborated to fight off extracellular pathogens.
Th2 cells are mediators of Ig production (humoral immunity) and
produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13 (Tanaka, et. al.,
Cytokine Regulation of Humoral Immunity, 251-272, Snapper, ed.,
John Wiley and Sons, New York (1996)). Th2-type immune responses
are characterized by the generation of certain cytokines (e.g.,
IL-4, IL-13) and specific types of antibodies (IgE, IgG4) and are
typical of allergic reactions, which may result in watery eyes and
asthmatic symptoms, such as airway inflammation and contraction of
airway muscle cells in the lungs.
In addition, the disorder that is preferably treated, ameliorated
or prevented by the methods of the present disclosure by applying
the lipocalins as described herein, may be associated with allergic
reaction or allergic inflammation.
In some preferred embodiments, the disorder may be allergic asthma,
rhinitis, conjunctivitis or dermatitis.
Asthma is a complex, persistent, inflammatory disease characterized
by airway hyper-responsiveness in association with airway
inflammation. Studies suggest that regular use of high-dose inhaled
corticosteroids and long-acting bronchodilators or omalizumab (a
humanized monoclonal antibody that binds to immunoglobulin E and is
often used as next-step therapy) may not be sufficient to provide
asthma control in all patients, highlighting an important unmet
need. Interleukin-4, interleukin-13, and the signal transducer and
activator of transcription factor-6 are key components in the
development of airway inflammation, mucus production, and airway
hyper-responsiveness in asthma. Biological compounds targeting
these molecules may provide a new therapeutic modality for patients
with uncontrolled moderate to severe asthma. The present disclosure
provides these biological compounds by way of the lipocalin muteins
as described herein.
In some preferred embodiments, the allergic asthma is an airway
inflammation in which the IL4/IL13 pathway contributes to disease
pathogenesis.
Furthermore, the disorder that is preferably treated, ameliorated
or prevented by the methods of the present disclosure by applying
the lipocalins as described herein, may also be lung disorders, for
example, pulmonary disorders in which the IL4/IL13 pathway
contributes to disease pathogenesis. Such pulmonary disorders
include but are not limited to, lung fibrosis, including chronic
fibrotic lung disease, other conditions characterized by
IL-4-induced fibroblast proliferation or collagen accumulation in
the lungs, pulmonary conditions in which a Th2 immune response
plays a role, conditions characterized by decreased barrier
function in the lung (e.g., resulting from IL-4-induced damage to
the epithelium), or conditions in which IL-4 plays a role in an
inflammatory response.
Similarly, Cystic fibrosis (CF) is characterized by the
overproduction of mucus and development of chronic infections.
Inhibiting IL-4RA and the Th2 response will reduce mucus production
and help control infections such as allergic bronchopulmonary
aspergillosis (ABPA). Allergic bronchopulmonary mycosis occurs
primarily in patients with cystic fibrosis or asthma, where a Th2
immune response is dominant. Inhibiting IL-4RA and the Th2 response
will help clear and control these infections.
Similarly, chronic obstructive pulmonary disease (COPD) is
associated with mucus hypersecretion and fibrosis. Inhibiting
IL-4RA and the Th2 response will reduce the production of mucus and
the development of fibrous thereby improving respiratory function
and delaying disease progression. Bleomycin-induced pneumopathy and
fibrosis, and radiation-induced pulmonary fibrosis are disorders
characterized by fibrosis of the lung which is manifested by the
influx of Th2, CD4.sup.+ cells and macrophages, which produce IL-4
and IL-13 which in turn mediates the development of fibrosis.
Inhibiting IL-4RA and the Th2 response will reduce or prevent the
development of these disorders.
Moreover, IL-4 and IL-13 induce the differentiation of lung
epithelial cells into mucus-producing goblet cells. IL-4 and IL-13
may therefore contribute to an enhanced production of mucus in
subpopulations or some situations. Mucus production and secretion
contributes to disease pathogenesis in chronic obstructive
pulmonary disease (COPD) and cystic fibrosis (CF). Thus, the
disorder, associated with a mucus production or a mucus secretion
(for example, overproduction or hypersecretion), can be preferably
treated, ameliorated or prevented by the methods of the present
disclosure by applying a lipocalin mutein as described herein. In
some preferred embodiments, the disorder, associated with a mucus
production or a mucus secretion is preferably a chronic obstructive
pulmonary disease (COPD) or a cystic fibrosis (CF). In other
preferred embodiments, the composition of the disclosure further
comprises an anti-mucus medicament.
Pulmonary alveolar proteinosis is characterized by the disruption
of surfactant clearance. IL-4 increases surfactant product. In some
further embodiments, use of an IL-4RA antagonist such as a
lipocalin mutein of the disclosure to decrease surfactant
production and decrease the need for whole lung lavage, is also
contemplated herein.
Adult respiratory distress syndrome (ARDS) may be attributable to a
number of factors, one of which is exposure to toxic chemicals.
Therefore, as a preferred but non-limiting example, one patient
population susceptible to ARDS is critically ill patients who go on
ventilators, as ARDS is a frequent complication in such patients.
In some further embodiments, an IL-4RA antagonist such as a
lipocalin mutein of the disclosure may thus be used to alleviate,
prevent or treat ARDS by reducing inflammation and adhesion
molecules.
Sarcoidosis is characterized by granulomatous lesions. In some
further embodiments, use of an IL-4RA antagonist such as a
lipocalin mutein of the disclosure to treat sarcoidosis,
particularly pulmonary sarcoidosis, is also contemplated
herein.
Conditions in which IL-4-induced barrier disruption plays a role
(e.g., conditions characterized by decreased epithelial barrier
function in the lung) may be treated with IL-4RA antagonist(s).
Damage to the epithelial barrier in the lungs may be induced by
IL-4 and/or IL-13 directly or indirectly. The epithelium in the
lung functions as a selective barrier that prevents contents of the
lung lumen from entering the submucosa. A damaged or "leaky"
barrier allows antigens to cross the barrier, which in turn elicits
an immune response that may cause further damage to lung tissue.
Such an immune response may include recruitment of eosinophils or
mast cells, for example. An IL-4RA antagonist may be administered
to inhibit such undesirable stimulation of an immune response.
In this regards, an IL-4RA antagonist such as a lipocalin mutein of
the disclosure may be employed to promote healing of lung
epithelium, in asthmatics for example, thus restoring barrier
function, or alternatively, administered for prophylactic purposes,
to prevent IL-4 and/or IL-13-induced damage to lung epithelium.
It should be noted that methods for treating a disease or disorder
in accordance with the present disclosure are not limited by a
particular mechanism of action. For example, in some further
embodiments, the lipocalin mutein of the disclosure may be utilized
as gene therapy agents in disclosed methods.
As used herein, "treatment" refers to clinical intervention in an
attempt to alter the natural course of the subject or cell being
treated, and can be performed before or during the course of
clinical pathology. Desirable effects of treatment include
preventing the occurrence or recurrence of a disease or a condition
or symptom thereof, alleviating a condition or symptom of the
disease, diminishing any direct or indirect pathological
consequences of the disease, decreasing the rate of disease
progression, ameliorating or palliating the disease state, and
achieving remission or improved prognosis. In some embodiments,
methods and compositions of the disclosure are useful in attempts
to delay development of a disease or disorder. The treatment is
applicable to both human therapy and veterinary applications.
"Prevention" includes that the disorders (or symptoms associated
therewith) described herein may be avoided before they occur and/or
that the disorders do not recur.
"Amelioration" includes that the disorders (or symptoms associated
therewith) described herein are alleviated, diminished, decreased
and/or palliated.
The term "administered" or "administering" in all of its
grammatical forms means administration of a therapeutically
effective dose of the lipocalin mutein as the sole therapeutic
agent or in combination with another therapeutic agent as described
herein to a subject. It is thus envisaged that a lipocalin mutein
of the present disclosure is preferably in the form of a
composition, preferably pharmaceutical composition, that may
preferably be employed in co-therapy approaches, i.e. in
co-administration with other medicaments or drugs, for example,
other medicaments for treating a disorder as described herein
and/or any other therapeutic agent which might be beneficial in the
context of the methods of the present disclosure. Thus, it is
preferred that a composition comprising the lipocalin muteins
described herein further comprises an anti-allergic medicament
and/or anti-allergic inflammation medicament.
An "effective amount", as used herein, refers to an amount
effective, at dosages and for periods of time necessary of a
lipocalin mutein as described herein, to achieve the desired
therapeutic or prophylactic result. The exact dose will depend on
the purpose of the treatment, and will be ascertainable by one
skilled in the art using known techniques.
By "therapeutically effective amount" it is meant a dose that
produces the effects for which it is administered. A
"therapeutically effective amount" of a lipocalin mutein as
described herein may vary according to factors such as age, body
weight, general health, sex, diet, time of administration, drug
interaction and the severity of the condition may be necessary, and
will be ascertainable with routine experimentation by those skilled
in the art. Sometimes, the term "therapeutically effective amount"
may sometimes be interchangeably used herein with the term
"pharmaceutically effective amount":
A therapeutically effective amount, when used in the present
application, is also one in which any toxic or detrimental effects
of the lipocalin mutein are outweighed by the therapeutically
beneficial effects. A "prophylactically effective amount" refers to
an amount effective, at dosages and for periods of time necessary
of a lipocalin mutein as described herein, to achieve the desired
prophylactic result. Typically but not necessarily, since a
prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be
less than the therapeutically effective amount. The lipocalin
mutein described herein having the desired therapeutic activity may
be administered in a physiologically acceptable carrier to a
patient, as described herein.
In some embodiments, a "subject", when used in the present
application, is a vertebrate. In certain particular embodiments,
the vertebrate is a mammal. Mammals include, but are not limited
to, primates (including human and non-human primates) and rodents
(e.g., mice and rats). In certain particular embodiments, a mammal
subject is a human. In some further embodiments, a human subject,
synonymous with an individual, is a particularly preferred subject.
In some still further embodiments, a human subject in need thereof,
when used in the present application, is a patient.
A protein of the disclosure can be a mutein of a lipocalin,
preferably a lipocalin selected from the group consisting of
retinol-binding protein (RBP), bilin-binding protein (BBP),
apolipoprotein D (APO D), neutrophil gelatinase associated
lipocalin (NGAL), tear lipocalin (TLPC),
.alpha..sub.2-microglobulin-related protein (A2m), 24p3/uterocalin
(24p3), von Ebners gland protein 1 (VEGP 1), von Ebners gland
protein 2 (VEGP 2), and Major allergen Can f1 precursor (ALL-1),
with human NGAL being a preferred lipocalin and human tear
lipocalin being a more preferred lipocalin. As used herein, a
"lipocalin" is defined as monomeric protein of approximately 18-20
kDA in weight, having a cylindrical .beta.-pleated sheet
supersecondary structural region comprising a plurality of
(preferably eight) .beta.-strands connected pair-wise by a
plurality of (preferably four) loops at one end to define thereby a
binding pocket. It is the diversity of the loops in the otherwise
rigid lipocalin scaffold that gives rise to a variety of different
binding modes among the lipocalin family members, each capable of
accommodating targets of different size, shape, and chemical
character (reviewed, e.g., in Skerra, A. (2000) Biochim. Biophys.
Acta 1482, 337-350). Indeed, the lipocalin family of proteins have
naturally evolved to bind a wide spectrum of ligands, sharing
unusually low levels of overall sequence conservation (often with
sequence identities of less than 20%) yet retaining a highly
conserved overall folding pattern. The correspondence between
positions in various lipocalins is well known to one of skill in
the art. See, for example, U.S. Pat. No. 7,250,297.
Generally when referred to herein the disclosure, a lipocalin
mutein of the disclosure, preferably, is different from its
naturally occurring counterpart lipocalin in that it differs in at
least one amino acid from its naturally occurring counterpart
lipocalin. The difference might be an amino acid substitution,
deletion and/or addition, with a substitution being preferred. In
certain particular embodiments, a "mutein of a lipocalin" or
"lipocalin mutein" can, in particular, be a "mutein of human tear
lipocalin" or "Tlc mutein".
In a preferred embodiment, a protein of the disclosure is a mutein
of human tear lipocalin (Tlc). The term "human tear lipocalin" as
used herein to refer to the mature human tear lipocalin with the
SWISS-PROT Data Bank Accession Number P31025. Mature human tear
lipocalin (amino acids 19-176 of SWISS-PROT Accession Number P31025
acid sequence) (SEQ ID NO: 1) does not include the N-terminal
signal peptide (amino acids 1-18) that is included in the sequence
of SWISS-PROT Accession Number P31025 acid sequence used as the
"reference" or "reference sequence" in various embodiments
described herein.
Human tear pre-albumin, now called tear lipocalin (TLPC or Tlc),
was originally described as a major protein of human tear fluid
(approximately one third of the total protein content) but has
recently also been identified in several other secretory tissues
including prostate, nasal mucosa and tracheal mucosa. Homologous
proteins have been found in rat, pig, dog and horse. Tear lipocalin
is an unusual lipocalin member because of its high promiscuity for
relative insoluble lipids and binding characteristics that differ
from other members of this protein family (reviewed in Redl, B.
(2000) Biochim. Biophys. Acta 1482, 241-248). A remarkable number
of lipophilic compounds of different chemical classes such as fatty
acids, fatty alcohols, phospholipids, glycolipids and cholesterol
are endogenous ligands of this protein. Interestingly, in contrast
to other lipocalins the strength of ligand (target) binding
correlates with the length of the hydrocarbon tail both for alkyl
amides and fatty acids. Thus, tear lipocalin binds most strongly
the least soluble lipids (Glasgow, B J. et al. (1995) Curr. Eye
Res. 14, 363-372; Gasymov, O. K. et al. (1999) Biochim. Biophys.
Acta 1433, 307-320).
Lipocalin muteins particularly suitable for a therapeutic use as
disclosed herein include the lipocalins set forth in SEQ ID NOs:
2-11. These molecules exhibit a specificity and high affinity for
human IL4Ra, but prior the present disclosure have not been
characterized with therapeutically relevant in vivo data. Other
lipocalin muteins that are also suitable for a therapeutic use as
disclosed herein include the lipocalins set forth in SEQ ID NOs:
2-8 of WO 2008/015239, each of these sequences is incorporated
herein by reference. Further lipocalin muteins that may also be
suitable for a therapeutic use as disclosed herein include the
lipocalins set forth in SEQ ID NOs: 2-11 of WO 2011/154420, each of
these sequences is incorporated herein by reference.
As used herein, a "mutein," a "mutated" entity (whether protein or
nucleic acid) or "mutant" refers to the exchange (substitution),
deletion, or insertion of one or more nucleotides or amino acids,
respectively, compared to the naturally occurring (wild-type)
nucleic acid or protein "reference" scaffold. A substitution of an
amino acid of the naturally occurring (wild-type) nucleic acid or
protein is preferred. The terms "reference", "reference sequence"
and "wild type sequence" are used interchangeably herein.
The present disclosure also contemplates optimized variants of the
lipocalin muteins specifically disclosed herein. Once a lipocalin
mutein with affinity to a given target has been selected, it is
possible to subject the mutein to further mutagenesis in order to
subsequently select variants of even higher affinity or variants
with improved properties such as higher thermostability, improved
serum stability, thermodynamic stability, improved solubility,
improved monomeric behavior, improved resistance against thermal
denaturation, chemical denaturation, proteolysis, or detergents
etc. This further mutagenesis, which in case of aiming at higher
affinity can be considered as in vitro "affinity maturation", can
be achieved by site specific mutation based on rational design or a
random mutation. Another possible approach for obtaining a higher
affinity or improved properties is the use of error-prone PCR,
which results in point mutations over a selected range of sequence
positions of the lipocalin mutein. The error-prone PCR can be
carried out in accordance with any known protocol such as the one
described by Zaccolo et al. (1996) J. Mol. Biol. 255, 589-603.
Other methods of random mutagenesis suitable for such purposes
include random insertion/deletion (RID) mutagenesis as described by
Murakami, H et al. (2002) Nat. Biotechnol. 20, 76-81 or
nonhomologous random recombination (NRR) as described by Bittker,
J. A et al. (2002) Nat. Biotechnol. 20, 1024-1029. If desired,
affinity maturation can also be carried out according to the
procedure described in WO 00/75308 or Schlehuber, S. et al., (2000)
J. Mol. Biol. 297, 1105-1120, where muteins of the bilin-binding
protein having high affinity to digoxigenin were obtained.
A tear lipocalin mutein may be used for complex formation with IL 4
receptor alpha. The mutein may also be able to bind an immunogenic
fragment of IL 4 receptor alpha. An immunogenic fragment of IL-4
receptor alpha is a fragment that has one or more epitopes,
mimotopes or other antigenic determinants, and is thus capable of
inducing an immune response or against which an antibody can be
raised. The immunogenic fragment may include a single epitope or
may have a plurality of epitopes. Since an antigen-presenting
system, e.g. a carrier protein, may be used to provide the size
required for recognition by an immune system, no particular size
limitation applies to the immunogenic fragment. Hence, the
immunogenic fragment may also be a "hapten", i.e. a fragment that
need not be antigenic per se or may have low immunogenicity, in
particular due to its small molecular weight and accordingly size.
Typically an immunogenic fragment can, alone or when presented on a
carrier, be bound by an immunoglobulin or by a T cell receptor
(TCR) if presented by MHC molecules. An immunogenic fragment is
typically, alone or when presented in the form of the
antigen-presenting system, capable of inducing a humoral immune
response and/or cellular immune response leading for instance to
the activation of B- and/or T-lymphocytes.
One target of the lipocalin mutein used in the present disclosure
is the alpha chain of the interleukin-4 receptor (officially known
as Interleukin-4 receptor subunit alpha, Uniprot #P24394, SEQ ID
NO: 12), a transmembrane protein, which contains an extracellular
domain of 207 amino acids. A secreted form of the extracellular
domain exists, sIL-4R alpha, which is also known as CD124 and
capable of blocking IL-4 activities. A lipocalin mutein of the
disclosure may be able to bind sIL-4 receptor alpha as well as any
portion of the extracellular domain of IL-4 receptor alpha.
The present disclosure also relates to nucleic acid molecules (DNA
and RNA) that include nucleotide sequences coding for lipocalin
muteins as described herein. Since the degeneracy of the genetic
code permits substitutions of certain codons by other codons
specifying the same amino acid, the disclosure is not limited to a
specific nucleic acid molecule encoding a lipocalin mutein of the
disclosure but encompasses all nucleic acid molecules that include
nucleotide sequences encoding a functional mutein.
The disclosed nucleic acid molecules can also be part of a vector
or any other kind of cloning vehicle, such as a plasmid, a
phagemid, a phage, a baculovirus, a cosmid or an artificial
chromosome. In one embodiment, the nucleic acid molecule is
included in a phasmid.
In some disclosed tear lipocalin muteins, the naturally occurring
disulfide bond between Cys 61 and Cys 153 is removed. Accordingly,
such muteins (or any other tear lipocalin mutein that does not
include an intramolecular disulfide bond) can be produced in a cell
compartment having a reducing redox milieu, for example, in the
cytoplasma of Gram-negative bacteria. In case a lipocalin mutein of
the disclosure includes intramolecular disulfide bonds, it may be
desired to direct the nascent polypeptide to a cell compartment
having an oxidizing redox milieu using an appropriate signal
sequence. Such an oxidizing environment may be provided by the
periplasm of Gram-negative bacteria such as E. coli, in the
extracellular milieu of Gram-positive bacteria or in the lumen of
the endoplasmatic reticulum of eukaryotic cells and usually favors
the formation of structural disulfide bonds. It is, however, also
possible to produce a lipocalin mutein of the disclosure in the
cytosol of a host cell, such as E. coli. In this case, the
polypeptide can either be directly obtained in a soluble and folded
state or recovered in form of inclusion bodies, followed by
renaturation in vitro. A further option is the use of specific host
strains having an oxidizing intracellular milieu, which may thus
allow the formation of disulfide bonds in the cytosol (Venturi M,
et al. (2002) J. Mol. Biol. 315, 1-8).
The disclosure also relates to a composition, which includes at
least one lipocalin mutein of the disclosure or a fragment or
variant thereof or a fusion protein or conjugate thereof, and a
pharmaceutically acceptable formulation (e.g. combination of
buffer/salt/excipient), wherein the said composition may be
administered to a subject in need thereof at a dosage level
selected from the group consisting of: 0.06-600 mg, 0.06-100 mg,
0.3-10 mg, 1-3 mg.
In some embodiments, a composition of the present disclosure can be
administered via any parenteral or non-parenteral (enteral) route
that is therapeutically effective for proteinaceous drugs.
Parenteral application methods include, for example,
intracutaneous, subcutaneous, intramuscular, intratracheal,
intranasal, intravitreal or intravenous injection and infusion
techniques, e.g. in the form of injection solutions, infusion
solutions or tinctures, as well as aerosol installation and
inhalation, e.g. in the form of aerosol mixtures, sprays, mist or
powders.
In some embodiments, local administration of the composition to the
lung is preferred. A preferred route of local administration to the
lung is via aerosol inhalation. An overview about pulmonary drug
delivery, i.e. either via inhalation of aerosols (which can also be
used in intranasal administration) or intratracheal instillation is
given by Patton, J. S., et al. (2004) Proc. Amer. Thoracic Soc., 1,
338-344, for example). Non-parenteral delivery modes are, for
instance, orally, e.g. in the form of pills, tablets, capsules,
solutions or suspensions, or rectally, e.g. in the form of
suppositories. In some embodiments, a lipocalin mutein of the
disclosure can also be administered systemically or topically in
formulations containing conventional non-toxic pharmaceutically
acceptable excipients or carriers, additives and vehicles as
desired. For administration of a lipocalin mutein of the disclosure
via inhalation, the mutein may be present in a powder formulation
together with an additional phase. The micronized powder of the
lipocalin mutein may be mixed with a carrier material, for example
lactose, dextrose, maltose, trehalose, as described in the patent
specification WO96/02231, or the article of P. Lucas, K. Anderson,
J. N. Staniforth, Protein deposition from dry powder inhalers,
Pharmaceutical Research, Vol. 15, April 1988, pages 562-569.
Alternatively, for inhalation purposes solid pharmaceutical
preparations as described in US patent applications 2005/0014677
and 2009/0142407 may be used.
Therefore, a composition of the disclosure may comprise a lipocalin
mutein of the disclosure as well as one of the disclosed
formulations.
The dosage of the composition applied may vary within wide limits
to achieve the desired preventive effect or therapeutic response.
It will, for instance, depend on the affinity of the lipocalin
mutein therein for a chosen ligand as well as on the half-life of
the complex between the lipocalin mutein and the ligand in vivo.
Further, the optimal dosage will depend on the biodistribution of
the lipocalin mutein or a fragment or variant thereof or its fusion
protein or its conjugate, the mode of administration, the severity
of the disease/disorder being treated as well as the medical
condition of the patient.
In some embodiments, the present disclosure provides a method of
treating a subject in need thereof, comprising administering to the
said subject a pharmaceutical composition via nebulization, wherein
the pharmaceutical composition comprising at least an IL-4RA
antagonist, or a fragment or variant thereof or a fusion protein or
conjugate thereof and a suitable solution.
In various preferred embodiments, a pharmaceutically acceptable
formulation of the disclosure can be a formulation for
nebulization, for example a solution that can maintain the
functional and structural integrity of the antagonist, or a
fragment or variant thereof or a fusion protein or conjugate
thereof, upon and after nebulization (e.g. remained monomeric and
fully functionally active) and/or, can, in combination with the
employed nebulizer, generates droplets of the desired size range.
In this regard, as an exemplary illustration, the present
disclosure teaches: the antagonist, for example, a lipocalin mutein
of the disclosure, can be formulated as a solution comprising, e.g.
0.01 mg to 100 mg of the antagonist or a fragment or variant
thereof or a fusion protein or conjugate thereof per 1 ml of a
saline having a pH adjusted to between 5.5 and 8.0, an osmolality
value between about 150 and 550 mOsm/kg, an ion concentration
between about 31 and 300 mM of chloride as a permeant anion, and/or
a viscosity smaller than 1.5 cp. Preferably, the formulated
solution has a concentration of the antagonist or a fragment or
variant thereof or a fusion protein or conjugate thereof at a level
selected from the group consisting of: 0.05 mg/ml to 50 mg/ml, 0.1
mg/ml to 50 mg/ml, 0.05 mg/ml to 25 mg/ml, 0.1 mg/ml to 25 mg/ml,
0.05 mg/ml to 15 mg/ml, 0.1 mg/ml to 15 mg/ml, 0.05 mg/ml to 10
mg/ml and 0.1 mg/ml to 10 mg/ml. In one further preferred
embodiment, the disclosed saline has an adjusted pH selected from
the group consisting of between 5.5 and 8.0, between 6.0 and 8.0,
between 6.0 and 7.5, between 5.5 and 7.5, between 6.0 and 7.0 and
between 6.5 and 7.5; and/or an osmolality value selected from the
group consisting of: between about 150 and 550 mOsm/L, between
about 150 and 500 mOsm/L, between about 200 and 550 mOsm/L, between
about 200 and 500 mOsm/L, between about 200 and 450 mOsm/L and
between about 150 and 450 mOsm/L, Osmolarity is the measure of
solute concentration, defined as the number of osmoles (Osm) of
solute per liter (L) of solution, e.g. osmol/L or Osm/L; and/or an
ion concentration of chloride as a permeant anion from the group
consisting of between 31 and 300 mM, between 50 and 200 mM, between
50 and 300 mM, between 50 and 150 mM, 100 and 200 mM, between 100
and 300 mM, between 100 and 250 mM, between 150 and 250 mM, between
150 and 300 mM and between 200 and 300 mM.
Further preferably, the disclosed saline has a viscosity selected
from the group consisting of between 0 and 1.5 cp, between 0 and
1.0 cp, 0 and 0.5 cp, between 0.5 and 1.0 cp, between 0.5 and 1.5
cp and between 1.0 and 1.5 cp.
In some further embodiments, the aqueous solution of the
antagonist, for example, a lipocalin mutein of the disclosure, can
be nebulized with an electronic nebulizer, jet, ultrasonic, or
vibrating perforated membrane, e.g. the PARI eFlow or Aeroneb
vibrating mesh nebulizer. In one preferred embodiment, the solution
is nebulized into an aerosol having a mass median aerodynamic
diameter (MMAD) between 1 .mu.m and 10 .mu.m, for electronic
nebulizer, jet, ultrasonic, or vibrating perforated membrane, 2
times daily, and the said nebulizer is able to aerosolize the
resulted solution into particles of required sizes (the aerosols)
in a time period from about 1 to about 5 minutes. In some still
further embodiment, any one of said aerosols may have a mass median
aerodynamic diameter (MMAD) selected from the group consisting of
between 1 .mu.m to 10 .mu.m, between 2 .mu.m to 8 .mu.m, between 2
.mu.m to 5 .mu.m, between 1.5 .mu.m to 4 .mu.m, between 3 .mu.m to
4.5 .mu.m and between 2.5 .mu.m to 3.5 .mu.m.
In some embodiments, the disclosure provides a method of delivery
by inhalation, within 1-3 minutes, preferably 1-2 minutes, of
efficacious amount of a pharmaceutical composition of the
disclosure, comprising a disclosed nebulization formulation, to a
subject in need thereof. In some further embodiments, said delivery
by inhalation is locally to the lung.
In some other embodiments, therefore, the disclosure provides an
efficacious, safe, nonirritating, physiologically acceptable and
compatible solution suitable for being used in a method of the
disclosure comprising administering a disclosed pharmaceutical
composition to a subject in need thereof.
In some other embodiments, the disclosure provides use the solution
for delivering a pharmaceutical composition of the disclosure to a
subject in need thereof as aerosol, for example, by inhalation.
When used herein, "IL-4RA antagonist" means a polypeptide or
protein that binds to IL-4RA with detectable binding affinity
and/or is capable of inhibiting IL-4 and/or IL-13 from binding to
their respective receptors. As used herein, "detectable affinity"
means the ability to bind to a selected target with an affinity
constant of generally at least about 10.sup.-5 M. Lower affinities
are generally no longer measurable with common methods such as
ELISA and therefore of secondary importance. For example, binding
affinities of muteins according to the disclosure may in some
embodiments be of a K.sub.D below 800 nM, in some embodiments be of
a K.sub.D below 30 nM and in some embodiments about 50 picomolar
(pM) or below
In some embodiments, a lipocalin mutein of the present disclosure
can be used to treat any disease or disorder in which the IL-4
expression and/or IL-13 expression contributes or is related to
disease pathogenesis or aggravation. In some embodiments, a
lipocalin mutein of the present disclosure can be used to treat any
disease or disorder that is improved, ameliorated or inhibited by
removal, inhibition or reduction of the IL-4 activity and/or IL-13
activity. As an illustrative example, the mutein or pharmaceutical
composition containing the same may be utilized in a method of
treating a disease or disorder associated with an increase of the
Th2 immune response. Such disease or disorder may, for example,
also be associated with an allergic reaction or an allergic
inflammation. In some further embodiments, such disease or disorder
may be allergic asthma, rhinitis, conjunctivitis or dermatitis (cf.
Hage et al. (1999) Cell, 97, 271-281, or Mueller et al. (2002)
Biochemica et Biophysica Acta, 237-250).
The term "fragment" as used in the present disclosure in connection
with the tear lipocalin muteins of the disclosure relates to
proteins or peptides derived from full-length mature human tear
lipocalin that are N-terminally and/or C-terminally shortened, i.e.
lacking at least one of the N-terminal and/or C-terminal amino
acids. Such fragments comprise preferably at least 10, more
preferably 20, most preferably 30 or more consecutive amino acids
of the primary sequence of mature human tear lipocalin and are
usually detectable in an immunoassay of mature human tear
lipocalin.
The term "variant" as used in the present disclosure relates to
derivatives of a protein or peptide (e.g. a tear lipocalin mutein
of the disclosure) that comprise modifications of the amino acid
sequence, for example by substitution, deletion, insertion or
chemical modification. Preferably, such modifications do not reduce
the functionality of the protein or peptide. Such variants include
proteins, wherein one or more amino acids have been replaced by
their respective D-stereoisomers or by amino acids other than the
naturally occurring 20 amino acids, such as, for example,
ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine,
norvaline. However, such substitutions may also be conservative,
i.e. an amino acid residue is replaced with a chemically similar
amino acid residue. Examples of conservative substitutions are the
replacements among the members of the following groups: 1) alanine,
serine, and threonine; 2) aspartic acid and glutamic acid; 3)
asparagine and glutamine; 4) arginine and lysine; 5) isoleucine,
leucine, methionine, and valine; and 6) phenylalanine, tyrosine,
and tryptophan.
The term "position" when used in accordance with the disclosure
means the position of either an amino acid within an amino acid
sequence depicted herein or the position of a nucleotide within a
nucleic acid sequence depicted herein. The term "corresponding" as
used herein also includes that a position is not only determined by
the number of the preceding nucleotides/amino acids. Accordingly,
the position of a given amino acid in accordance with the
disclosure which may be substituted may very due to deletion or
addition of amino acids elsewhere in a (mutant or wild-type)
lipocalin. Similarly, the position of a given nucleotide in
accordance with the present disclosure which may be substituted may
vary due to deletions or additional nucleotides elsewhere in a
mutein or wild type lipocalin 5'-untranslated region (UTR)
including the promoter and/or any other regulatory sequences or
gene (including exons and introns). Thus, under a "corresponding
position" in accordance with the disclosure it is preferably to be
understood that nucleotides/amino acids may differ in the indicated
number but may still have similar neighbouring nucleotides/amino
acids. Said nucleotides/amino acids which may be exchanged, deleted
or added are also comprised by the term "corresponding position".
Specifically, in order to determine whether an amino acid residue
of the amino acid sequence of a lipocalin (mutein) different from a
wild-type lipocalin corresponds to a certain position in the amino
acid sequence of a wild-type lipocalin, a skilled artisan can use
means and methods well-known in the art, e.g., alignments, either
manually or by using computer programs such as BLAST2.0, which
stands for Basic Local Alignment Search Tool or ClustalW or any
other suitable program which is suitable to generate sequence
alignments. Accordingly, a wild-type lipocalin can serve as
"subject sequence" or "reference sequence", while the amino acid
sequence of a lipocalin different from the wild-type lipocalin
described herein serves as "query sequence". The terms "reference
sequence" and "wild type sequence" are used interchangeably
herein.
As used herein, the singular forms "a", "an", and "the", include
plural references unless the context clearly indicates otherwise.
Thus, for example, reference to "a lipocalin mutein" includes one
or more lipocalin muteins.
Unless otherwise indicated, the term "at least" preceding a series
of elements is to be understood to refer to every element in the
series. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the disclosure described
herein. Such equivalents are intended to be encompassed by the
present disclosure.
Throughout this specification and the claims which follow, unless
the context requires otherwise, the word "comprise", and variations
such as "comprises" and "comprising", will be understood to imply
the inclusion of a stated integer or step or group of integers or
steps but not the exclusion of any other integer or step or group
of integer or step. When used herein the term "comprising" can be
substituted with the term "containing" or "having".
When used herein "consisting of" excludes any element, step, or
ingredient not specified in the claim element. When used herein,
"consisting essentially of" does not exclude materials or steps
that do not materially affect the basic and novel characteristics
of the claim.
As used herein, the conjunctive term "and/or" between multiple
recited elements is understood as encompassing both individual and
combined options. For instance, where two elements are conjoined by
"and/or", a first option refers to the applicability of the first
element without the second. A second option refers to the
applicability of the second element without the first. A third
option refers to the applicability of the first and second elements
together. Any one of these options is understood to fall within the
meaning, and therefore satisfy the requirement of the term "and/or"
as used herein. Concurrent applicability of more than one of the
options is also understood to fall within the meaning, and
therefore satisfy the requirement of the term "and/or" as used
herein.
Several documents are cited throughout the text of this
specification. Each of the documents cited herein (including all
patents, patent applications, scientific publications,
manufacturer's specifications, instructions, etc.), whether supra
or infra, are hereby incorporated by reference in their entirety.
Nothing herein is to be construed as an admission that the
disclosure is not entitled to antedate such disclosure by virtue of
prior disclosure.
EXAMPLES
Example 1
Inhibition of IL-4 and IL-13 Induced Stat6 Phosphorylation in TF-1
Cells by an IL-4 Receptor Alpha Antagonist
TF-1 cells were incubated at 37.degree. C. for 30 min with an IL-4
receptor alpha antagonist, a lipocalin mutein directed against the
human IL-4 receptor alpha chain (SEQ ID NO: 6), before adding 10 nM
IL-13 (FIG. 1a) or 0.1 nM IL-4 (FIG. 1b). Cells were fixed after
incubation for 15 min at 37.degree. C. with 1.6% PFA and
permeabilized with 100% methanol prior to intracellular STAT6
staining with PE Mouse Anti-STAT6 (pY641)-BD 612701. The IL-4RA
specific antibody MAB230 (R&D Systems) and the IL-4 double
mutant IL-4 (R121D, Y124D) as described by Andrews et al. JI 2006
176:7456-7461 was used as positive control. In addition, the human
tear lipocalin (SEQ ID NO: 1) and a mouse IgG2a antibody (mIgG2a,
Ancell 281-010; Lot#171605) were used as negative controls. The
results from the TF-1 Stat6 phosphorylation assay are depicted in
FIG. 1 and show that the lipocalin mutein (SEQ ID NO: 6) is a
potent antagonists of IL-4 as well as IL-13 as it relates to IL-4RA
downstream signaling. The IL-4RA specific antibody MAB230 (R&D
Systems) performs equally well. The IL-4 mutant to our surprise is
significantly less effective in inhibiting both IL-4 and IL-13
induced Stat6 phosphorylation in TF-1 cells although functional
antagonism had been described previously. The fast dissociation
rate constant (k.sub.off) of the IL-4 mutant (R121D, Y124D)
compared to the lipocalin mutein (Table 1) might be the reason for
this functional difference.
TABLE-US-00001 TABLE 1 Test substance KD k.sub.on k.sub.off
Lipocalin Mutein 23 pM 4.1 .times. 10.sup.6 M.sup.-1s.sup.-1 9.2
.times. 10.sup.-5 s.sup.-1 (SEQ ID NO: 6) IL-4 mutant 154 pM 1.9
.times. 10.sup.7 M.sup.-1s.sup.-1 2.9 .times. 10.sup.-3 s.sup.-1
(R121D, Y124D)
Example 2
Construction of Double Knock-in Mouse
Human IL-4 receptor alpha chain and IL-13 receptor alpha chain 1
knock-in mice were generated by replacing the mouse IL-4 receptor
alpha chain gene and the IL-13 receptor alpha chain 1 gene with the
respective human orthologue. Accordingly, the generated mice are
so-called human IL-4 receptor alpha/human IL-13 receptor alpha
chain 1 double knock-in mice which harbor the human instead of the
mouse IL-4 receptor alpha and IL-13 receptor alpha chain 1 genes at
the mouse orthologous gene locus.
The mice were generated as follows: The 70 kb mouse IL-13RA1 gene
on chromosome X was deleted, generating a null allele in mouse
embryonic stem cells. A segment of DNA carrying the human IL-13RA1
gene was introduced into the deleted locus. The human gene consists
of a 95.7 kb fragment of the human X chromosome that contains not
only the entire IL13RA gene but also .about.11 kb of 5' inter-genic
DNA and .about.7 kb of DNA 3' of the 3'UTR. Implicit in this is the
knowledge that this fragment of DNA will direct the expression of
the human receptor. Mice carrying this alteration were generated
from the modified ES cells. In a separate experiment, the 39 kb
segment of DNA carrying the mouse IL-4RA gene was exchanged with
the corresponding segment of 63 kb of DNA encoding human IL-4R,
which is composed of IL-4R alpha and the common gamma chain, and
again using a mouse line generated from the modified cells. The two
Knock-in mouse lines were intercrossed to establish a mouse line
homozygous for the humanized locus at both the IL-4R locus and the
IL-13RA locus. The mice express the human proteins in the absence
of the mouse proteins. The proper expression of the human Type I
and Type II receptors was verified by demonstrating the response of
immune cells to human IL4 and the response of airway epithelial
cells to IL-13.
Example 3
Ex Vivo Analysis of Double Knock-in Mouse Splenocytes to Verify the
Function of an IL-4 Receptor Alpha Antagonist on the IL-4 Type I
Receptor
Splenocytes were isolated from human IL-4 receptor alpha/human
IL-13 receptor alpha chain 1 double knock-in mice by means and
methods commonly known in the art. These splenocytes were
stimulated with increasing concentrations of human IL-4 either in
the absence or presence of an IL-4 receptor alpha antagonist, a
lipocalin mutein directed against the human IL-4 receptor alpha
chain (SEQ ID NO: 6). IL-4 effects phosphorylation of STAT6 via the
IL-4 receptor complex. Thus, phosphorylation of STAT6 is therefore
a read-out for IL-4 binding to its receptor. The human tear
lipocalin (SEQ ID NO: 1) serves as a negative control.
In FIG. 2, it is shown that the lipocalin mutein (SEQ ID NO: 6)
decreases, and thus neutralizes, action of IL-4 on the IL-4 type I
receptor expressed on splenocytes, since STAT 6 phosphorylation in
IL-4 stimulated splenocytes was equal to the level of unstimulated
splenocytes.
Example 4
Administration of an IL-4 Receptor Alpha Antagonist to Double
Knock-in Transgenic Mouse & Analysis of Eotaxin Levels
The IL-13 induced airway inflammation model provided by Blanchard
et al. (Clin Exp Allergy 2005, 35(8):1096-1103 was used to
investigate the effect of an IL-4 receptor alpha antagonist, a
lipocalin mutein directed against human IL-4 receptor alpha chain.
In the model, 1 .mu.g IL-13 is administered three times every 48
hours by intratracheal instillation.
Accordingly, as shown in FIG. 3, vehicle (phosphate buffered
saline, thereafter referred as "PBS") or IL-13 (hIL-13) was
administered to double knock-in transgenic mice, as group 1 (PBS)
and group 2 (hIL-13), respectively, whereas group 3 (SEQ ID NO:
6/hIL-13) and group 4 (TLPC/hIL-13) received the lipocalin mutein
(SEQ ID NO: 6) at a dose of 57 .mu.g or the human tear lipocalin
(SEQ ID NO: 1) as negative control at a dose of 57 .mu.g,
respectively, in addition to having received hIL-13.
Groups 2 and 3 contained 3 mice, while groups 1 and 4 contained 2
mice. Intratracheal dosing was done at 30 .mu.l. IL-13, the
lipocalin mutein (SEQ ID NO: 6) and the negative control were
administered intratracheally and lung tissue homogenate was
obtained with the aim of measuring eotaxin levels. Note that the
lipocalin mutein and the negative control were administered 30
minutes prior to IL-13 administration.
When IL-13 is administered in this model, eotaxin levels increase
e.g. 24 hours after the last IL-13 dose in group 4 compared to
group1. Eotaxin is a potent eosinophil chemoattractant and thus a
marker for inflammation, in particular allergic inflammation. It is
shown in FIG. 3 that the lipocalin mutein (SEQ ID NO: 6) in group 4
effectively neutralizes the action of IL-13 on eotaxin induction in
comparison to the negative control in group 3 (also cf. PBS group 1
and IL-13 alone group 2).
Example 5
Administration of an IL-4 Receptor Alpha Antagonist to Double
Knock-in Transgenic Mouse & Analysis of mRNA Expression of
Ccl11 (Eotaxin)
Administration of an IL-4 receptor alpha antagonist, a lipocalin
mutein directed against human IL-4 receptor alpha chain (SEQ ID NO:
6), to human IL-4 receptor alpha/human IL-13 receptor alpha chain 1
double knock-in mice was done as described in Example 4 with the
exception that 30 .mu.l of IL-13 (Peprotech, 1 .mu.g) was
administered by intratracheal instillation only once. Each of the
groups contained 3 human IL-4 receptor alpha/human IL-13 receptor
alpha chain 1 double knock-in mice. Total RNA was isolated from
lung homogenates 24 hours after the IL-13 dose and analyzed for
mouse eotaxin expression by RT-PCR. mRNA expression in lung tissue
after 24 h are normalized with 18S rRNA and the value for the 1
.mu.g hIL13 mice set as 1. The lipocalin mutein (SEQ ID NO: 6) was
also applied by intratracheal instillation with a volume of 30
.mu.l either at a constant dose of 98 .mu.g at different times
prior to the IL-13 dose or at different amounts 30 minutes prior to
the IL-13 dose. The IL-4 mutant (R121D, Y124D) was also applied by
intratracheal instillation with a volume of 30 .mu.l at different
amounts 30 minutes prior to the IL-13 dose. The abbreviated IL-13
induced airway inflammation model (single IL-13 intratracheal
administration) was used to assess the duration of the
pharmacological response, dose dependency and comparable potency.
In FIG. 4a, it can be seen that the lipocalin mutein (SEQ ID NO: 6)
inhibited human IL-13 induced transcript Ccl11 (eotaxin)
effectively for an extended period of time (FIG. 4a), while dose
dependency of the pharmacological response was also demonstrated as
shown in FIG. 4b. In addition, as can be seen in FIG. 4c, a
favorable potency of the lipocalin mutein (SEQ ID NO: 6) was shown
when compared side by side with the IL-4 mutant (FIG. 4c).
Example 6
Identification of Formulations Suitable for Nebulizing an IL-4
Receptor Alpha Antagonist Using the Pari eFlow Vibrating Mesh
Nebulizer
An IL-4 receptor alpha antagonist, an IL-4RA-specific lipocalin
mutein (SEQ ID NO: 6), was nebulized with a Pari eFlow device at a
concentration of 0.1 mg/ml in PBS containing either 0.01 or 0.05
polysorbate 20. The reservoir of the nebulizer was filled with 6 ml
of the formulation and operated for approximately 15 minutes until
half of the reservoir was nebulized. A shown in FIG. 5, the
nebulized sample was collected with a glass vial fitted to the
mouth piece of the nebulizer and analyzed by visual inspection to
monitor visible particles, by light obscuration to monitor
subvisible particles, by high pressure size exclusion
chromatography (HP-SEC) to monitor soluble aggregates, by reverse
phase SEC to monitor chemical modifications, by an IL-4RA binding
ELISA to monitor functional activity and by laser diffraction to
measure droplet size. Nebulization of the lipocalin mutein (SEQ ID
NO: 6) in the chosen formulation did not lead to the generation of
visible aggregate, subvisible particles. Both the nebulized protein
and the protein remaining in the reservoir stayed monomeric and
fully functionally active. Droplets with a median size of 5.5 .mu.m
were generated according to the specifications of the device. The
concentration of polysorbate 20 in the formulation did not seem to
influence the ability to nebulizer the mutein as none of the
measured parameters were identical in formulations containing 0.01
or 0.05 polysorbate 20.
Example 7
Pharmacokinetic Properties and Biodistribution of an IL-4 Receptor
Alpha Antagonist in Human IL-4RA/Human IL-13 Double Knock-in Mice
after Intratracheal Instillation
An IL-4 receptor alpha antagonist, the lipocalin mutein (SEQ ID NO:
6) was administered by intratracheal administration of 30 .mu.l (98
.mu.g) to human IL-4 receptor alpha/human IL-13 receptor alpha
chain 1 double knock-in mice. Twenty animals were dosed and groups
of 4 were sacrificed after 1, 4, 8, 16 and 24 hours. At the time of
sacrifice, lungs were lavaged with 2.times.1 ml FACSflow fluid,
Li-heparin plasma samples were drawn and the lungs were snap frozen
in liquid nitrogen. The lung tissue was homogenated in 1.5 ml lysis
buffer (one tablet of complete mini protease inhibitor cocktail
tablets (Roche)/10 ml T-PER (Perbio)) using an IKA T10 basic
Ultra-Turrax (S10N-5G) tissue homogenizer at 4.degree. C. for 55
seconds. The homogenate was incubated on ice for 30-60 minutes and
cleared by centrifugation with 10.000 g at 4.degree. C. for 10
minutes. Total protein concentration was determined with a BCA kit
(Pierce) according to the manufacturer's instructions and samples
were diluted with PBS to a standard final protein concentration of
1 mg/ml prior to storage of aliquots at -80.degree. C. A
quantitative Meso Scale Discovery ("MSD") based ELISA was used to
measure the binding active concentrations of the lipocalin mutein
(SEQ ID NO: 6) in lavage fluid, lung tissue and plasma at the
different time points. Briefly, MSD plates were coated with
neutravidin, 5 .mu.g/ml in PBS over night at 4.degree. C., washed
with PBS/0.05% Tween20 and blocked with 3% BSA. Biotinylated human
IL-4RA at a concentration of 2.5 .mu.g/ml in PBS/0.1% Tween20 was
added to capture the lipocalin mutein from the different matrixes
and the bound mutein was detected with a tear lipocalin specific
rabbit polyclonal antibody (preparation: TLPC-Mix4666 Ab, Pieris,
PL#684) and an anti-rabbit IgG Sulfo-Tag antibody (Meso Scale
Discovery, 0.5 .mu.g/ml) in PBS/0.5% Tween20/0.5% BSA. The assay
exhibited in all three matrixes a linear range from 75 pg/ml to 6
ng/ml while QC samples had a recovery between 80-120%. For
calculations of concentrations and total amounts a total plasma
volume of 900 .mu.l per mouse was assumed. The dilution factor used
to standardize the lung homogenate to 1 mg/ml total protein
concentration measured by the BCA kit was taking into account when
the total amount of lipocalin mutein per lung was calculated based
on the measured concentration in the standardized total lung
homogenate and its volume of 1.5 ml lysis buffer and measured
weight of the lungs. The total amount of the lipocalin mutein in
bronchoalveolar lavage fluid (thereafter "BALF") was based on the
amount recovered in 2 ml lavage fluid.
It can be seen in FIG. 6a that 43% of the administered dose could
be recovered in the BALF, 6% in the lung tissue and 0.2% in plasma
1 hour after intratracheal administration. A terminal half-life of
3.7, 3.9 and 2.7 hours was determined in the BALF, lung tissue and
plasma, respectively. Furthermore, it can be seen in FIG. 6b that
the calculated lung tissue concentrations in human IL-4RA/human
IL-13 double knock-in mice were maintained above 0.7 .mu.g/ml after
intratracheal instillation of 98 .mu.g of the lipocalin mutein for
approximately 20 hours which is in line with the observations shown
in FIG. 4a. The 0.7 .mu.g/ml concentration corresponds to the
highest IC.sub.90 value of the lipocalin mutein observed in
different in vitro potency assays. Furthermore, FIG. 6b also shows
that the plasma concentrations and therefore systemic exposure were
100 fold lower compared to the concentrations seen in the lung
tissue.
Example 8
Inhibition of IL-13 Induced Goblet Cell Metaplasia in Human Airway
Epithelium Air-Liquid-Interface Culture System by an IL-4 Receptor
Alpha Antagonist
Goblet cell metaplasia is a common feature of several respiratory
diseases including Asthma. Therefore, the ability of an IL-4
receptor alpha antagonist, the lipocalin mutein (SEQ ID NO: 6), to
prevent goblet cell metaplasia in an in vitro model based on
MucilAir.TM. (Epithelix) was assessed. MucilAir.TM., an air-liquid
interface culture system with human airway epithelium reconstituted
in vitro using primary human cells was treated every two days with
human IL-13 at 0.3 to 30 ng/ml. By in-situ Alcian blue staining, as
well as histological analysis, it was demonstrated that
MucilAir.TM. showed an increased goblet cell density after 14 days
of treatment, in a dose dependent manner. Therefore, the inhibitory
effect of the lipocalin mutein on goblet cell metaplasia was tested
by comparing continuous exposure of MucilAir.TM. to 10 ng/ml human
IL-13 for 14 days as positive control and to IL-13+ different
concentrations (as shown in FIG. 7a) of the lipocalin mutein (SEQ
ID NO: 6), the IL-4 mutant (R121D, Y124D) and an anti-IL-4RA
monoclonal antibody (the light and heavy chain variable region are
shown in SEQ D NOs: 14 and 15), respectively, and when compared to
MucilAir.TM. cultured for 14 days without IL-13 as negative
control. The Alcain blue stain, which stains acid
mucopolysacharides and glycosaminoglycans in blue to bluish-green,
was added to the apical surface for an in-situ stain and pictures
from stained cells were taken under a phase contrast microscope for
image analysis. The percentage of the Alcain blue positive cells
was quantified by the public domain Java image processing program
ImageJ and expressed as area ratio of Alcain blue area/total image
area. Histological analysis has been performed by a platform at the
Geneva University according to standard protocol. In addition,
eotaxin-3, an IL-13 induced chemokine, was measured in the basal
medium on day 14 using a commercially available ultrasensitive
eotaxin-3 Kit from Meso Scale Discovery according to the
manufacturer's instruction.
In FIG. 7a and FIG. 7b, it can be seen that the lipocalin mutein
(SEQ ID NO: 6) was able to block IL-13 induced goblet cell
metaplasia in the MucilAir culture system completely in a dose
dependent manner. The level of Alcain blue area ratio was reduced
to background (the ratio of the negative control having no IL-13)
by the lipocalin mutein as well as the anti-IL-4RA monoclonal
antibody but not by the IL-4 mutant (R121D, Y124D), while the human
tear lipocalin (SEQ ID NO: 1) exhibited similar staining as the
positive control (only having IL-13). Similar results were obtained
for eotaxin-3 (secreted into the basal culture media) as seen in
FIG. 7d. Exemplary Alcain blue/Neutral red stained paraffin
sections of day 14 cultures with and without IL-13 as shown in FIG.
7c displayed the impact of IL-13 on human airway epithelium in the
air-liquid-interface culture system.
The invention has industrial applications in connection with
treatment of diseases and/or conditions in which the IL4/IL13
pathway contributes to disease pathogenesis, e.g. diseases and/or
conditions associated with an increase of the Th2 immune response.
The invention illustratively described herein may suitably be
practiced in the absence of any element or elements, limitation or
limitations, not specifically disclosed herein. Thus, for example,
the terms "comprising", "including", "containing", etc. shall be
read expansively and without limitation. Additionally, the terms
and expressions employed herein have been used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof, but it is
recognized that various modifications are possible within the scope
of the invention claimed. Thus, it should be understood that
although the present invention has been specifically disclosed by
preferred embodiments and optional features, modification and
variation of the inventions embodied therein herein disclosed may
be resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope
of this invention. The invention has been described broadly and
generically herein. All patents, patent applications, text books
and peer-reviewed publications described herein are hereby
incorporated by reference in their entirety. Furthermore, where a
definition or use of a term in a reference, which is incorporated
by reference herein is inconsistent or contrary to the definition
of that term provided herein, the definition of that term provided
herein applies and the definition of that term in the reference
does not apply. Each of the narrower species and subgeneric
groupings falling within the generic disclosure also form part of
the invention. This includes the generic description of the
invention with a proviso or negative limitation removing any
subject matter from the genus, regardless of whether or not the
excised material is specifically recited herein. In addition, where
features or aspects of the invention are described in terms of
Markush groups, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual
member or subgroup of members of the Markush group. Further
embodiments of the invention will become apparent from the
following claims.
SEQUENCE LISTINGS
1
151158PRTHomo sapiensmature human tear lipocalin 1His His Leu Leu
Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr1 5 10 15 Trp Tyr
Leu Lys Ala Met Thr Val Asp Arg Glu Phe Pro Glu Met Asn 20 25 30
Leu Glu Ser Val Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn 35
40 45 Leu Glu Ala Lys Val Thr Met Leu Ile Ser Gly Arg Cys Gln Glu
Val 50 55 60 Lys Ala Val Leu Glu Lys Thr Asp Glu Pro Gly Lys Tyr
Thr Ala Asp65 70 75 80 Gly Gly Lys His Val Ala Tyr Ile Ile Arg Ser
His Val Lys Asp His 85 90 95 Tyr Ile Phe Tyr Cys Glu Gly Glu Leu
His Gly Lys Pro Val Arg Gly 100 105 110 Val Lys Leu Val Gly Arg Asp
Pro Lys Asn Asn Leu Glu Ala Leu Glu 115 120 125 Asp Phe Glu Lys Ala
Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile 130 135 140 Leu Ile Pro
Arg Gln Ser Glu Thr Cys Ser Pro Gly Ser Asp 145 150 155
2158PRTArtificialMutein 1 of human tear lipocalin with binding
affinity for IL-4R alpha 2His His Leu Leu Ala Ser Asp Glu Glu Ile
Gln Asp Val Ser Gly Thr 1 5 10 15 Trp Tyr Leu Lys Ala Met Thr Val
Asp Ser Arg Cys Pro Arg Ala Tyr 20 25 30 Tyr Ser Ser Val Thr Pro
Met Thr Leu Thr Thr Leu Glu Gly Gly Asn 35 40 45 Leu Glu Ala Lys
Phe Thr Ala Gln Arg Ser Gly Arg Trp Gln Glu Tyr 50 55 60 Lys Leu
Val Leu Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Ser 65 70 75 80
Gly Gly Arg His Val Ala Tyr Ile Ile Arg Ser His Val Lys Asp His 85
90 95 Tyr Ile Phe His Ser Glu Gly Leu Cys Pro Gly Gln Pro Val Pro
Gly 100 105 110 Val Trp Leu Val Gly Arg Asp Pro Lys Asn Asn Leu Glu
Ala Leu Glu 115 120 125 Asp Phe Glu Lys Ala Ala Gly Ala Arg Gly Leu
Ser Thr Glu Ser Ile 130 135 140 Leu Ile Pro Arg Gln Ser Glu Thr Ser
Ser Pro Gly Ser Ala 145 150 1553158PRTArtificialMutein 2 of human
tear lipocalin with binding affinity for IL-4R alpha 3His His Leu
Leu Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr 1 5 10 15 Trp
Tyr Leu Lys Ala Met Thr Val Asp Ser Arg Cys Pro Arg Ala Tyr 20 25
30 Tyr Glu Ser Val Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn
35 40 45 Leu Glu Ala Lys Leu Thr Leu Gln Arg Lys Gly Arg Trp Gln
Glu Met 50 55 60 Lys Asp Val Leu Glu Lys Thr Asp Glu Pro Gly Lys
Tyr Thr Ala Ser 65 70 75 80 Gly Gly Arg His Val Ala Tyr Ile Ile Arg
Ser His Val Lys Asp His 85 90 95 Tyr Ile Phe His Ser Glu Gly Leu
Cys Pro Gly Gln Pro Val Pro Gly 100 105 110 Val Trp Leu Val Gly Arg
Asp Pro Lys Asn Asn Leu Glu Ala Leu Glu 115 120 125 Asp Phe Glu Lys
Ala Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile 130 135 140 Leu Ile
Pro Arg Gln Ser Glu Thr Ser Ser Pro Gly Ser Ala 145 150
1554158PRTArtificialMutein 3 of human tear lipocalin with binding
affinity for IL-4R alpha 4His His Leu Leu Ala Ser Asp Glu Glu Ile
Gln Asp Val Ser Gly Thr 1 5 10 15 Trp Tyr Leu Lys Ala Met Thr Val
Asp Pro Arg Cys Pro Arg Ala Tyr 20 25 30 Tyr Ser Ser Val Thr Pro
Met Thr Leu Thr Thr Leu Glu Gly Gly Asn 35 40 45 Leu Glu Ala Lys
Phe Thr Ala Gln Arg Ser Gly Arg Trp Gln Lys Tyr 50 55 60 Lys Leu
Val Leu Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Ser 65 70 75 80
Gly Gly Arg His Val Ala Tyr Ile Ile Arg Ser His Val Lys Asp His 85
90 95 Tyr Ile Phe His Ser Glu Gly Leu Cys Pro Gly Gln Pro Val Pro
Gly 100 105 110 Val Trp Leu Val Gly Arg Asp Pro Lys Asn Asn Leu Glu
Ala Leu Glu 115 120 125 Asp Phe Glu Lys Ala Ala Gly Ala Arg Gly Leu
Ser Thr Glu Ser Ile 130 135 140 Leu Ile Pro Arg Gln Ser Glu Thr Ser
Ser Pro Gly Ser Ala 145 150 1555158PRTArtificialMutein 4 of human
tear lipocalin with binding affinity for IL-4R alpha 5His His Leu
Leu Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr 1 5 10 15 Trp
Tyr Leu Lys Ala Met Thr Val Asp Leu Arg Cys Pro Arg Ala Tyr 20 25
30 Tyr Trp Ser Val Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn
35 40 45 Leu Glu Ala Lys Phe Thr Ala Leu Arg Ile Gly Arg Trp Gln
Ser Tyr 50 55 60 Lys Leu Val Leu Glu Lys Thr Asp Glu Pro Gly Lys
Tyr Thr Ala Ser 65 70 75 80 Gly Gly Arg His Val Ala Tyr Ile Ile Arg
Ser His Val Lys Asp His 85 90 95 Tyr Ile Phe His Ser Glu Gly Leu
Cys Pro Gly Gln Pro Val Pro Gly 100 105 110 Val Trp Leu Val Gly Arg
Asp Pro Lys Asn Asn Leu Glu Ala Leu Glu 115 120 125 Asp Phe Glu Lys
Ala Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile 130 135 140 Leu Ile
Pro Arg Gln Ser Glu Thr Ser Ser Pro Gly Ser Ala 145 150
1556158PRTArtificialMutein 5 of human tear lipocalin with binding
affinity for IL-4R alpha 6His His Leu Leu Ala Ser Asp Glu Glu Ile
Gln Asp Val Ser Gly Thr 1 5 10 15 Trp Tyr Leu Lys Ala Met Thr Val
Asp Ser Arg Cys Pro Arg Ala Val 20 25 30 Tyr Asn Ser Val Thr Pro
Met Thr Leu Thr Thr Leu Glu Gly Gly Asn 35 40 45 Leu Glu Ala Lys
Phe Thr Ala Gln Arg Lys Gly Arg Trp Gln Lys Tyr 50 55 60 Lys Leu
Val Leu Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Ser 65 70 75 80
Gly Gly Arg His Val Ala Tyr Ile Ile Arg Ser His Val Lys Asp His 85
90 95 Tyr Ile Phe His Ser Glu Gly Leu Cys Pro Gly Gln Pro Val Pro
Gly 100 105 110 Val Trp Leu Val Gly Arg Asp Pro Lys Asn Asn Leu Glu
Ala Leu Glu 115 120 125 Asp Phe Glu Lys Ala Ala Gly Ala Arg Gly Leu
Ser Thr Glu Ser Ile 130 135 140 Leu Ile Pro Arg Gln Ser Glu Thr Ser
Ser Pro Gly Ser Ala 145 150 1557158PRTArtificialMutein 6 of human
tear lipocalin with binding affinity for IL-4R alpha 7His His Leu
Leu Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr 1 5 10 15 Trp
Tyr Leu Lys Ala Met Thr Val Asp Ser Arg Cys Pro Arg Ala Tyr 20 25
30 Tyr Val Ser Val Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn
35 40 45 Leu Glu Ala Lys Phe Thr Ala Ala Arg Ile Gly Arg Trp Gln
Ser Tyr 50 55 60 Lys Leu Val Leu Glu Lys Thr Asp Glu Pro Gly Lys
Tyr Thr Ala Ser 65 70 75 80 Gly Gly Arg His Val Ala Tyr Ile Ile Arg
Ser His Val Lys Asp His 85 90 95 Tyr Ile Phe His Ser Glu Gly Leu
Cys Pro Gly Gln Pro Val Pro Gly 100 105 110 Val Trp Leu Val Gly Arg
Asp Pro Lys Asn Asn Leu Glu Ala Leu Glu 115 120 125 Asp Phe Glu Lys
Ala Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile 130 135 140 Leu Ile
Pro Arg Gln Ser Glu Thr Ser Ser Pro Gly Ser Ala 145 150
1558158PRTArtificialMutein 7 of human tear lipocalin with binding
affinity for IL-4R alpha 8His His Leu Leu Ala Ser Asp Glu Glu Ile
Gln Asp Val Ser Gly Thr 1 5 10 15 Trp Tyr Leu Lys Ala Met Thr Val
Asp Asn Arg Cys Pro Arg Ala Lys 20 25 30 Tyr Asp Ser Val Thr Pro
Met Thr Leu Thr Thr Leu Glu Gly Gly Asn 35 40 45 Leu Glu Ala Lys
Phe Thr Ala His Arg Arg Gly Arg Trp Gln Gln Tyr 50 55 60 Lys Leu
Val Leu Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Ser 65 70 75 80
Gly Gly Arg His Val Ala Tyr Ile Ile Arg Ser His Val Lys Asp His 85
90 95 Tyr Ile Phe His Ser Glu Gly Leu Cys Pro Gly Gln Pro Val Pro
Gly 100 105 110 Val Trp Leu Val Gly Arg Asp Pro Lys Asn Asn Leu Glu
Ala Leu Glu 115 120 125 Asp Phe Glu Lys Ala Ala Gly Ala Arg Gly Leu
Ser Thr Glu Ser Ile 130 135 140 Leu Ile Pro Arg Gln Ser Glu Thr Ser
Ser Pro Gly Ser Ala 145 150 1559158PRTArtificialMutein 8 of human
tear lipocalin with binding affinity for IL-4R alpha 9His His Leu
Leu Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr 1 5 10 15 Trp
Tyr Leu Lys Ala Met Thr Val Asp Tyr Arg Cys Pro Arg Ala Tyr 20 25
30 Tyr His Ser Val Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn
35 40 45 Leu Glu Ala Lys Phe Thr Ala His Arg Ala Gly Arg Trp Gln
Lys Tyr 50 55 60 Lys Leu Val Leu Glu Lys Thr Asp Glu Pro Gly Lys
Tyr Thr Ala Ser 65 70 75 80 Gly Gly Arg His Val Ala Tyr Ile Ile Arg
Ser His Val Lys Asp His 85 90 95 Tyr Ile Phe His Ser Glu Gly Leu
Cys Pro Gly Gln Pro Val Pro Gly 100 105 110 Val Trp Leu Val Gly Arg
Asp Pro Lys Asn Asn Leu Glu Ala Leu Glu 115 120 125 Asp Phe Glu Lys
Ala Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile 130 135 140 Leu Ile
Pro Arg Gln Ser Glu Thr Ser Ser Pro Gly Ser Ala 145 150
15510158PRTArtificialMutein 9 of human tear lipocalin with binding
affinity for IL-4R alpha 10His His Leu Leu Ala Ser Asp Glu Glu Ile
Gln Asp Val Ser Gly Thr 1 5 10 15 Trp Tyr Leu Lys Ala Met Thr Val
Asp Lys Arg Cys Pro Arg Ala Tyr 20 25 30 Tyr Arg Ser Val Thr Pro
Met Thr Leu Thr Thr Leu Glu Gly Gly Asn 35 40 45 Leu Glu Ala Lys
Phe Thr Ala Lys Arg Asn Gly Arg Trp Gln Pro Tyr 50 55 60 Lys Leu
Val Leu Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Ser 65 70 75 80
Gly Gly Arg His Val Ala Tyr Ile Ile Arg Ser His Val Lys Asp His 85
90 95 Tyr Ile Phe His Ser Glu Gly Leu Cys Pro Gly Gln Pro Val Pro
Gly 100 105 110 Val Trp Leu Val Gly Arg Asp Pro Lys Asn Asn Leu Glu
Ala Leu Glu 115 120 125 Asp Phe Glu Lys Ala Ala Gly Ala Arg Gly Leu
Ser Thr Glu Ser Ile 130 135 140 Leu Ile Pro Arg Gln Ser Glu Thr Ser
Ser Pro Gly Ser Ala 145 150 15511158PRTArtificialMutein 10 of human
tear lipocalin with binding affinity for IL-4R alpha 11His His Leu
Leu Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr 1 5 10 15 Trp
Tyr Leu Lys Ala Met Thr Val Asp Glu Arg Cys Pro Arg Ala His 20 25
30 Tyr Gly Ser Val Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn
35 40 45 Leu Glu Ala Lys Phe Thr Ala Met Arg Leu Gly Arg Trp Gln
Lys Tyr 50 55 60 Lys Leu Val Leu Glu Lys Thr Asp Glu Pro Gly Lys
Tyr Thr Ala Ser 65 70 75 80 Gly Gly Arg His Val Ala Tyr Ile Ile Arg
Ser His Val Lys Asp His 85 90 95 Tyr Ile Phe His Ser Glu Gly Leu
Cys Pro Gly Gln Pro Val Pro Gly 100 105 110 Val Trp Leu Val Gly Arg
Asp Pro Lys Asn Asn Leu Glu Ala Leu Glu 115 120 125 Asp Phe Glu Lys
Ala Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile 130 135 140 Leu Ile
Pro Arg Gln Ser Glu Thr Ser Ser Pro Gly Ser Ala 145 150
15512825PRTHomo sapiensHuman IL-4 receptor 12Met Gly Trp Leu Cys
Ser Gly Leu Leu Phe Pro Val Ser Cys Leu Val 1 5 10 15 Leu Leu Gln
Val Ala Ser Ser Gly Asn Met Lys Val Leu Gln Glu Pro 20 25 30 Thr
Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu Trp Lys Met 35 40
45 Asn Gly Pro Thr Asn Cys Ser Thr Glu Leu Arg Leu Leu Tyr Gln Leu
50 55 60 Val Phe Leu Leu Ser Glu Ala His Thr Cys Ile Pro Glu Asn
Asn Gly 65 70 75 80 Gly Ala Gly Cys Val Cys His Leu Leu Met Asp Asp
Val Val Ser Ala 85 90 95 Asp Asn Tyr Thr Leu Asp Leu Trp Ala Gly
Gln Gln Leu Leu Trp Lys 100 105 110 Gly Ser Phe Lys Pro Ser Glu His
Val Lys Pro Arg Ala Pro Gly Asn 115 120 125 Leu Thr Val His Thr Asn
Val Ser Asp Thr Leu Leu Leu Thr Trp Ser 130 135 140 Asn Pro Tyr Pro
Pro Asp Asn Tyr Leu Tyr Asn His Leu Thr Tyr Ala 145 150 155 160 Val
Asn Ile Trp Ser Glu Asn Asp Pro Ala Asp Phe Arg Ile Tyr Asn 165 170
175 Val Thr Tyr Leu Glu Pro Ser Leu Arg Ile Ala Ala Ser Thr Leu Lys
180 185 190 Ser Gly Ile Ser Tyr Arg Ala Arg Val Arg Ala Trp Ala Gln
Cys Tyr 195 200 205 Asn Thr Thr Trp Ser Glu Trp Ser Pro Ser Thr Lys
Trp His Asn Ser 210 215 220 Tyr Arg Glu Pro Phe Glu Gln His Leu Leu
Leu Gly Val Ser Val Ser 225 230 235 240 Cys Ile Val Ile Leu Ala Val
Cys Leu Leu Cys Tyr Val Ser Ile Thr 245 250 255 Lys Ile Lys Lys Glu
Trp Trp Asp Gln Ile Pro Asn Pro Ala Arg Ser 260 265 270 Arg Leu Val
Ala Ile Ile Ile Gln Asp Ala Gln Gly Ser Gln Trp Glu 275 280 285 Lys
Arg Ser Arg Gly Gln Glu Pro Ala Lys Cys Pro His Trp Lys Asn 290 295
300 Cys Leu Thr Lys Leu Leu Pro Cys Phe Leu Glu His Asn Met Lys Arg
305 310 315 320 Asp Glu Asp Pro His Lys Ala Ala Lys Glu Met Pro Phe
Gln Gly Ser 325 330 335 Gly Lys Ser Ala Trp Cys Pro Val Glu Ile Ser
Lys Thr Val Leu Trp 340 345 350 Pro Glu Ser Ile Ser Val Val Arg Cys
Val Glu Leu Phe Glu Ala Pro 355 360 365 Val Glu Cys Glu Glu Glu Glu
Glu Val Glu Glu Glu Lys Gly Ser Phe 370 375 380 Cys Ala Ser Pro Glu
Ser Ser Arg Asp Asp Phe Gln Glu Gly Arg Glu 385 390 395 400 Gly Ile
Val Ala Arg Leu Thr Glu Ser Leu Phe Leu Asp Leu Leu Gly 405 410 415
Glu Glu Asn Gly Gly Phe Cys Gln Gln Asp Met Gly Glu Ser Cys Leu 420
425 430 Leu Pro Pro Ser Gly Ser Thr Ser Ala His Met Pro Trp Asp Glu
Phe 435 440 445
Pro Ser Ala Gly Pro Lys Glu Ala Pro Pro Trp Gly Lys Glu Gln Pro 450
455 460 Leu His Leu Glu Pro Ser Pro Pro Ala Ser Pro Thr Gln Ser Pro
Asp 465 470 475 480 Asn Leu Thr Cys Thr Glu Thr Pro Leu Val Ile Ala
Gly Asn Pro Ala 485 490 495 Tyr Arg Ser Phe Ser Asn Ser Leu Ser Gln
Ser Pro Cys Pro Arg Glu 500 505 510 Leu Gly Pro Asp Pro Leu Leu Ala
Arg His Leu Glu Glu Val Glu Pro 515 520 525 Glu Met Pro Cys Val Pro
Gln Leu Ser Glu Pro Thr Thr Val Pro Gln 530 535 540 Pro Glu Pro Glu
Thr Trp Glu Gln Ile Leu Arg Arg Asn Val Leu Gln 545 550 555 560 His
Gly Ala Ala Ala Ala Pro Val Ser Ala Pro Thr Ser Gly Tyr Gln 565 570
575 Glu Phe Val His Ala Val Glu Gln Gly Gly Thr Gln Ala Ser Ala Val
580 585 590 Val Gly Leu Gly Pro Pro Gly Glu Ala Gly Tyr Lys Ala Phe
Ser Ser 595 600 605 Leu Leu Ala Ser Ser Ala Val Ser Pro Glu Lys Cys
Gly Phe Gly Ala 610 615 620 Ser Ser Gly Glu Glu Gly Tyr Lys Pro Phe
Gln Asp Leu Ile Pro Gly 625 630 635 640 Cys Pro Gly Asp Pro Ala Pro
Val Pro Val Pro Leu Phe Thr Phe Gly 645 650 655 Leu Asp Arg Glu Pro
Pro Arg Ser Pro Gln Ser Ser His Leu Pro Ser 660 665 670 Ser Ser Pro
Glu His Leu Gly Leu Glu Pro Gly Glu Lys Val Glu Asp 675 680 685 Met
Pro Lys Pro Pro Leu Pro Gln Glu Gln Ala Thr Asp Pro Leu Val 690 695
700 Asp Ser Leu Gly Ser Gly Ile Val Tyr Ser Ala Leu Thr Cys His Leu
705 710 715 720 Cys Gly His Leu Lys Gln Cys His Gly Gln Glu Asp Gly
Gly Gln Thr 725 730 735 Pro Val Met Ala Ser Pro Cys Cys Gly Cys Cys
Cys Gly Asp Arg Ser 740 745 750 Ser Pro Pro Thr Thr Pro Leu Arg Ala
Pro Asp Pro Ser Pro Gly Gly 755 760 765 Val Pro Leu Glu Ala Ser Leu
Cys Pro Ala Ser Leu Ala Pro Ser Gly 770 775 780 Ile Ser Glu Lys Ser
Lys Ser Ser Ser Ser Phe His Pro Ala Pro Gly 785 790 795 800 Asn Ala
Gln Ser Ser Ser Gln Thr Pro Lys Ile Val Asn Phe Val Ser 805 810 815
Val Gly Pro Thr Tyr Met Arg Val Ser 820 825 13427PRTHomo
sapienshuman IL-13 receptor alpha 1 13Met Glu Trp Pro Ala Arg Leu
Cys Gly Leu Trp Ala Leu Leu Leu Cys 1 5 10 15 Ala Gly Gly Gly Gly
Gly Gly Gly Gly Ala Ala Pro Thr Glu Thr Gln 20 25 30 Pro Pro Val
Thr Asn Leu Ser Val Ser Val Glu Asn Leu Cys Thr Val 35 40 45 Ile
Trp Thr Trp Asn Pro Pro Glu Gly Ala Ser Ser Asn Cys Ser Leu 50 55
60 Trp Tyr Phe Ser His Phe Gly Asp Lys Gln Asp Lys Lys Ile Ala Pro
65 70 75 80 Glu Thr Arg Arg Ser Ile Glu Val Pro Leu Asn Glu Arg Ile
Cys Leu 85 90 95 Gln Val Gly Ser Gln Cys Ser Thr Asn Glu Ser Glu
Lys Pro Ser Ile 100 105 110 Leu Val Glu Lys Cys Ile Ser Pro Pro Glu
Gly Asp Pro Glu Ser Ala 115 120 125 Val Thr Glu Leu Gln Cys Ile Trp
His Asn Leu Ser Tyr Met Lys Cys 130 135 140 Ser Trp Leu Pro Gly Arg
Asn Thr Ser Pro Asp Thr Asn Tyr Thr Leu 145 150 155 160 Tyr Tyr Trp
His Arg Ser Leu Glu Lys Ile His Gln Cys Glu Asn Ile 165 170 175 Phe
Arg Glu Gly Gln Tyr Phe Gly Cys Ser Phe Asp Leu Thr Lys Val 180 185
190 Lys Asp Ser Ser Phe Glu Gln His Ser Val Gln Ile Met Val Lys Asp
195 200 205 Asn Ala Gly Lys Ile Lys Pro Ser Phe Asn Ile Val Pro Leu
Thr Ser 210 215 220 Arg Val Lys Pro Asp Pro Pro His Ile Lys Asn Leu
Ser Phe His Asn 225 230 235 240 Asp Asp Leu Tyr Val Gln Trp Glu Asn
Pro Gln Asn Phe Ile Ser Arg 245 250 255 Cys Leu Phe Tyr Glu Val Glu
Val Asn Asn Ser Gln Thr Glu Thr His 260 265 270 Asn Val Phe Tyr Val
Gln Glu Ala Lys Cys Glu Asn Pro Glu Phe Glu 275 280 285 Arg Asn Val
Glu Asn Thr Ser Cys Phe Met Val Pro Gly Val Leu Pro 290 295 300 Asp
Thr Leu Asn Thr Val Arg Ile Arg Val Lys Thr Asn Lys Leu Cys 305 310
315 320 Tyr Glu Asp Asp Lys Leu Trp Ser Asn Trp Ser Gln Glu Met Ser
Ile 325 330 335 Gly Lys Lys Arg Asn Ser Thr Leu Tyr Ile Thr Met Leu
Leu Ile Val 340 345 350 Pro Val Ile Val Ala Gly Ala Ile Ile Val Leu
Leu Leu Tyr Leu Lys 355 360 365 Arg Leu Lys Ile Ile Ile Phe Pro Pro
Ile Pro Asp Pro Gly Lys Ile 370 375 380 Phe Lys Glu Met Phe Gly Asp
Gln Asn Asp Asp Thr Leu His Trp Lys 385 390 395 400 Lys Tyr Asp Ile
Tyr Glu Lys Gln Thr Lys Glu Glu Thr Asp Ser Val 405 410 415 Val Leu
Ile Glu Asn Leu Lys Lys Ala Ser Gln 420 425
14109PRThumanmisc_feature(1)..(109)12B5 antibody variable light
chain from WO 01/92340 14Glu Ile Val Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Phe Gly Ala
Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85
90 95 Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
15115PRThumanmisc_feature(1)..(115)12B5 antibody variable heavy
chain from WO 01/92340 15Glu Val Gln Leu Val Gln Ser Gly Gly Gly
Leu Val His Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly
Ser Gly Phe Thr Phe Ser Arg Asn 20 25 30 Ala Met Phe Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Gly
Thr Gly Gly Ala Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala 85
90 95 Arg Gly Arg Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110 Val Ser Ser 115
* * * * *